GRN has recently been associated with a risk of frontotemporal lobar degeneration (FTLD) in North American population particularly in pathologically confirmed cases with neural inclusions immunoreactive for ubiquitin and TAR DNA-binding protein 43 (TDP-43), but negative for tau and alpha-synuclein (FTLD-TDP).A single nucleotide polymorphism (rs5848) located in the 3′- untranslated region of In an effort to replicate these results in a different population, rs5848 was genotyped in 256 FTLD cases and 1695 controls from the Netherlands. Single SNP gender-adjusted logistic regression analysis revealed no significant association between variation at rs5848 and FTLD. Fisher's exact test, failed to find any significant association between rs5848 and a subset of 23 pathology confirmed FTLD-TDP cases.The evidence presented here suggests that variation at rs5848 does not contribute to the etiology of FTLD in the Dutch population. Frontotemporal lobar degeneration (FTLD) is the second most common form of presenile dementia after Alzheimer's disease (AD), representing ∼5% of all dementia cases and as much as 10–20% of presenile dementia FTLD is characterized by not specific symptoms such as apathy and lack of interest, usually followed by specific features such as disinhibition or progressive language loss. These symptoms gradually evolve to cognitive impairment and dementia.Pathologically, FTLD is characterized by neural loss and gliosis in the cortical lamina II of the frontal and temporal lobes. FTLD is pathologically heterogeneous and can be divided in FTLD with tau-positive inclusions (FTLD-tau), and FTLD with neural inclusions immunoreactive for ubiquitin and TAR DNA-binding protein 43 (TDP-43), but negative for tau and alpha-synuclein (FTLD-TDP) GRN) are a major cause of FTLD-TDP worldwide counting for as much as 25% of familiar FTLD-TDP FTLD has a high familial incidence with up to 50% of patients reported to have a family history of similar dementia, parkinsonism or MND GRN is significantly associated with the development of FTLD, particularly in a homogeneous series of patients with a neuropathological diagnosis of FTLD-TDP GRN. They successfully demonstrate that miR-659 binds more efficiently to the GRN 3′-UTR containing the risk T-allele of rs5848 resulting in a drop of GRN protein levels. As expected, no changes were detected in the GRN mRNA levels.Recently, Rademakers and colleagues demonstrated that a single nucleotide polymorphism (rs5848) located in the 3′- untranslated region (3′-UTR) of In an effort to replicate the results obtained by Rademakers and collaborators and to confirm the role of rs5848 in modulating the risk for FTLD, this SNP was genotyped in a large series of 287 FTLD cases  and 1695 controls from the Netherlands Approval was given by the Medical Ethical Committee of the Erasmus University, Rotterdam, the Netherlands; after written informed consent of the patients that their information could be stored in the hospital database and used for research.A total of 287 patients with FTD were ascertained from an ongoing genetic-epidemiologic study conducted in the Netherlands since 1994 after referral to the out-clinic department of the Erasmus Medical Center, or after visiting nursing homes and psychogeriatric hospitals by the research physician The age at onset was defined as the age at which the first symptom, compatible with FTD diagnosis, was observed by a close relative or caretaker. During the neurological examination carried out in all patients, special attention was paid to the presence of extrapyramidal and motor neuron disease signs.MAPT and GRN genes, those in which an autopsy did not confirm FTLD pathology, those without a Caucasian ancestry and those closely related to some other sample in our dataset, where removed from further analysis. Thus, our final FTLD cohort consisted of 256 cases of which 120 where males and 136 females. The main age at onset was 57.73 years ranging from 28.64 to 75.95 years (SD = 8.94).After genotyping, all samples presenting with mutations in http://www.lasa-vu.nl/) with a mean age of 68.78 years at the time of medical examination .Control samples consisted of 805 males and 890 females from the Longitudinal Aging Study Amsterdam genotypicP = 0.29).Although we are aware that the age distribution of this cohort cannot be matched to that from the FTLD cases, a single SNP sex-adjusted linear regression analysis in these samples considering the age at examination as a quantitative phenotype showed that genotypes at rs5848 are equally distributed across age (www.appliedbiosystems.com) were utilized to genotype rs5848 in all samples previously described. Primer and probes were design with the File Builder v3.1 software (www.appliedbiosystems.com) and genotyping was performed in a LightCycler 480 II PCR system using LightCycler 480 Probes Master mix (www.roche.com) as per manufacturer's instructions. For each reaction, 20 ng of genomic DNA was utilized.Custom TaqMan® MGB probes and primers from Applied Biosystems (http://pngu.mgh.harvard.edu/~purcell/plink) All tests and estimates were performed using the PLINK toolset for whole-genome analysis  revealed a normal distribution of the data.Statistical significance for differences in age at onset was tested using a gender-adjusted linear regression analysis and considering age at onset as a quantitative trait. Before performing this analysis, R v.2.7.2 software .Finally, in an effort to test whether rs5848 may have an influence on the age at onset of FTLD in our cohort, a gender-adjusted linear regression analysis following a recessive model was performed considering age at onset as an alternative quantitative phenotype. No association was detected after this analysis in the entire cases cohort  and 934 controls ascertained through the Mayo Clinic at Jacksonville and Scottsdale Pgenotypic = 0.002). Analysis of variation at this locus in our Dutch cohort showed no significant differences between the frequency of homozygous T carriers in cases versus controls (Precessive = 0.57). Although we cannot rule out a minimal contribution of this locus on modeling a risk for FTLD, the sample size of the population utilized for our statistical tests (256 FTLD cases and 1645 controls) provides an adequate power to detect an effect of the magnitude originally described (OR = 2.9 for T/T carriers versus T/C and C/C carriers). Thus, the evidence presented here suggests that variation at rs5848 does not contribute to the etiology of FTLD in the Dutch population.Here we present the analysis of rs5848 in a large cohort of FTLD cases and controls from the Netherlands. Variation at this We also failed to replicate Rademakers and collaborators' results showing an association between rs5848 and a subset of neuropathologically confirmed FTLD-TDP cases from the Mayo Clinic brain bank et al., who established a classification scheme of FTLD-TDP cases based on the distribution of the neuronal cytoplasmatic inclusions, dystrophic neurites and the presence of neuronal intranuclear inclusions P = 0.26).As proposed by Mackenzie GRN gene (including rs5848) in a Finnish population with AD Recently, Viswanathan and collaborators investigated the role of six different SNPs in the locus ranges across populations from 0.16 to 0.77 (for HapMap-CEU and HapMap YRI respectively) shows the susceptibility of this SNP to be affected by case-control admixture and, thus, lead to type I errors. Thus, results observed by Rademakers' and collaborators may represent a slant in the selection of the FTLD-TDP patients.In a similar report as that presented herein, Rollinson and collaborators also failed to find any association between rs5848 an FTLD in 3 different European populations In summary we conclude that variation at rs5848 does not affect the risk for developing FTLD in European populations. Although a small effect in the FTLD-TDP pathological subtype of the disease cannot be discarded, evidence presented here and elsewhere does not support this idea. Further genotyping of different populations of these and other characteristics is needed for better understanding the role of rs5848 on the pathogenesis of FTLD.


We examined whether candidate gene polymorphisms were associated with altered urine inflammatory profiles in asymptomatic women with or without bacteriuria.Although several studies suggest that genetic factors are associated with human UTI susceptibility, the role of DNA variation in regulating early We conducted a cross-sectional analysis of asymptomatic bacteriuria (ASB) in 1,261 asymptomatic women ages 18-49 years originally enrolled as participants in a population-based case-control study of recurrent UTI and pyelonephritis. We genotyped polymorphisms in CXCR1, CXCR2, TLR1, TLR2, TLR4, TLR5, and TIRAP in women with and without ASB. We collected urine samples and measured levels of uropathogenic bacteria, neutrophils, and chemokines.Polymorphism TLR2_G2258A, a variant associated with decreased lipopeptide-induced signaling, was associated with increased ASB risk . Three CXCR1 polymorphisms were associated with ASB caused by gram-positive organisms. ASB was associated with urinary CXCL-8 levels, but not CXCL-5, CXCL-6, or sICAM-1 (P≤0.0001). Urinary levels of CXCL-8 and CXCL-6, but not ICAM-1, were associated with higher neutrophil levels (P≤0.0001). In addition, polymorphism CXCR1_G827C was associated with increased CXCL-8 levels in women with ASB (P = 0.004).in vivo human bladder immune responses prior to the development of symptomatic UTIs.TLR2 and CXCR1 polymorphisms were associated with ASB and a CXCR1 variant was associated with urine CXCL-8 levels. These results suggest that genetic factors are associated with early  Asymptomatic bacteriuria provides an opportunity to investigate the role of genetic factors in the early phases of the human 3 cfu/mL of a urinary pathogen) or UTI guideline-related treatment was required for all UTI episodes in the cluster. Women experiencing previous pyelonephritis (n = 400) were identified through having received a pyelonephritis ICD-9 diagnosis code and, if they received only outpatient treatment, a primary diagnosis of pyelonephritis and an accompanying culture result of ≥103 cfu/mL of a urinary pathogen or accompanying antibiotic therapy appropriate for pyelonephritis. The remainder of the women constituted the potential control subjects (n = 430) with no history of UTIs who were randomly selected and frequency-matched by case age group . For these analyses, the 1261 participants, all asymptomatic at their study clinic visits, were classified into groups of ASB status based on the urine bacterial counts.The study protocols were approved by the Human Subjects committees at Group Health Cooperative, the University of Washington, and Western Institutional Review Board. The study was conducted at Group Health Cooperative in Seattle, Washington. Written informed consent was obtained from all subjects. We performed a cross-sectional study to examine whether candidate gene polymorphisms are associated with ASB or urine inflammatory markers in 1261 women. The subjects were enrolled as part of a case-control study of rUTI and pyelonephritis that has been previously described 3 cfu/ml were identified and quantified and E. coli were characterized as beta- or non-hemolytic. A second urine sample was collected in a sterile container to measure urinary neutrophil levels with a dipstick assay (Bayer Multistix Reagent Strips) and reported on a scale of negative, trace, small (+1), moderate (+2), or large (+3). The trace category was excluded from the analysis. The negative category was compared to the 3 other values. In addition, 2 mls of urine from the 2nd collection were immediately frozen and stored at −70°C for later testing for IL-6, CXCL-8, CXCL-5, CXCL-6, and sICAM-1 levels. Prior to testing, specimens were centrifuged at 12,000 RPM for 2 minutes, and the supernatants were removed for analysis. The supernatant was subjected to a sandwich ELISA technique . Values below the lowest detectable limit of the assay were replaced as zero .Subjects were asymptomatic at the time of their clinic visits. We collected a clean-catch midstream urine specimen in a sterile BD Vacutainer™ Urine Tube  containing a lyophilized preservative including boric acid and sodium formate. Specimens were stored at room temperature and transported to the laboratory within 72 hours where they were inoculated onto blood agar, MacConkey, and CNA plates. Plates were incubated at 37°C for 48 hours, and organisms were quantified in colony-forming units (cfu)/ml and further identified using standard methods http://innateimmunity.net/). For determining haplotype tagging SNPs, we selected polymorphisms in the chromosomal region of each gene which represented the commonly inherited haplotypes . Selection criteria were based on linkage disequilibrium measurements between the polymorphisms TM technique (Sequenom) as previously described Genomic DNA was purified from peripheral blood by QIAamp DNA blood kit (Qiagen). For genotyping in the full cohort, we generated haplotype-tagging SNPs from our sequencing data as well as publicly available data from the Innate Immunity Program in Genomic Applications , medium ASB (≥103 and <105 CFU/ml of uropathogen) and high ASB (≥105 CFU/ml of uropathogen). We also evaluated ASB as an exposure with urine neutrophils or chemokines as the outcomes. For these analyses, only cultures with uropathogens were examined. Cultures with non-uropathogens  were not included in the definition of ASB. Furthermore, any culture with greater than or equal to 100,000 CFU/ml of coagulase-negative staphylococci, mixed gram-negative flora, or mixed gram-positive flora was excluded from the analysis to avoid samples that may have been contaminated with skin or perineal flora during collection . Gram-negative uropathogens included E. coli non-hemolytic, E. coli β-hemolytic, Proteus species, Enterobacter species, Klebsiella species, Citrobacter species, and Morganella morganii. Gram-positive uropathogens included Enterococcus species, Lactobacillus species, Group B Streptococcus, Staphylococcus aureus, and S. saprophyticus. In addition to stratifying by ASB level, we performed subgroup analyses of ASB caused by gram-negative and gram-positive pathogens.Several different exposures and outcomes were assessed in the analysis. For the primary analysis, polymorphisms of candidate genes were classified as exposures and ASB or urine inflammatory markers (chemokines or neutrophils) were treated as the outcomes. To evaluate the association of genetic polymorphisms with ASB, we defined 3 outcome groups: no ASB  and ASB status or (the outcomes) were examined with an allelic trend test. In the allelic trend test  common homozygous genotypes (00) were assigned a value of 0, heterozygotes (01) a value of 1, and minor homozygous genotypes (11) a value of 2. Odds ratios and statistical significance values were then assessed using logistic regression models. We analyzed the ASB groups separately (medium ASB vs no ASB or high ASB vs no ASB) and as a combined group (medium + high ASB vs no ASB). Two-sided testing was used for all comparisons to evaluate statistical significance, considering a P-value of ≤0.05 as significant. To verify that our significant findings were not due to population admixture, we also performed Caucasian subgroup analyses. All analyses were performed using the software Hplus or SAS Univariate analyses were performed for categorical variables with a Chi-Square test; Fisher's exact test was used when the number of samples in a group was less than 5. The Mann-Whitney U-test was used to make comparisons of the chemokine levels between ASB Groups. We expressed the chemokine data as 75th or 90th percentile values due to the high number of women with undetectable urinary chemokines. The percent of women with undetectable chemokine levels was: 32.6% for CXCL-8, 39.1% for CXCL-5, 39.5% for CXCL-6, and 5.8% for sICAM-1. Chemokine levels were analyzed by genotype for each polymorphism with an allelic trend test using a general linear model.While these data were analyzed cross-sectionally, the study sample was originally selected for a study employing a case-control methodology, and thus case-control status could potentially confound the associations of current interest. To address this issue, we adjusted our analyses for case-control status, finding that this design variable did not act as a confounder. We also incorporated an interaction term for case-control status and the exposures of interest, to assess possible heterogeneity in associations.3 cfu/ml), medium ASB (≥103 to <105 cfu/ml), or high ASB (≥105 cfu/ml). The ethnic backgrounds of women in these 3 groups were similar with a predominance of a Caucasian background  and CXCR1 is associated with ASB caused by gram-positive uropathogens.th percentile value 55.7 pg/ml) vs. medium ASB (102.6 pg/ml) vs. high ASB (249.3 pg/ml). This association was present for ASB caused by both gram-negative as well as gram-positive pathogens. We also examined CXCL-5, CXCL-6, and sICAM-1 and found no difference in these outcomes by womens' ASB status. sICAM-1, in contrast to the other chemokines, had high basal levels in women without ASB. Together, these results suggest a selective association of ASB with CXCL-8, but not CXCL-5, CXCL-6, or ICAM-1.To examine whether ASB is associated with chemokine levels, we measured urine levels of CXCL-8, CXCL-5, CXCL-6, and sICAM-1. We also examined IL-6, but only a small number of women had detectable IL-6 levels , so no further measurement or analysis was performed. CXCL-8 was associated with increasing levels of ASB  in comparison to genotypes GC or CC   was associated with CXCL-8 concentration in women with ASB from all uropathogens (ctively) . We alsoctively) . CXCR2_ZIn this manuscript, we investigated the variation and genetic regulation of inflammation associated with asymptomatic bacteriuria in women in order to gain insight into the early stages of human UTI pathogenesis. Our main findings are that polymorphisms in TLR2 and CXCR1 are associated with ASB and that a CXCR1 polymorphism is associated with urine CXCL-8 levels in women with ASB. In addition, we found that chemokine levels are selectively associated with ASB  and urinary neutrophil levels  with ASB. Even though CXCL-8, CXCL-5, and CXCL-6 are ligands for CXCR1 and CXCR2, our data suggest differential regulation of these chemokines in response to bacteriuria. With at least 8 chemokines involved in neutrophil chemotaxis, it is often difficult to elucidate specific and/or non-redundant roles of individual chemokines. By focusing on ASB, we attempted to discover which chemokines are involved with neutrophil migration during early steps in UTI pathogenesis. With progression of disease to a symptomatic stage, a different pattern of elevated chemokines likely occurs. Previous studies of urine chemokine levels have mainly focused on IL-6 and CXCL-8 during episodes of symptomatic cystitis We also found a selective association of chemokines  with urine neutrophil levels. Previous studies demonstrated that urine neutrophil counts are associated with elevated CXCL-8 levels We also found an association of several CXCR1 SNPs  with ASB caused by gram-positive uropathogens. Furthermore, we found an association of polymorphism G827C in CXCR1 with CXCL-8 levels in women with ASB. This polymorphism , which encodes a serine to threonine substitution at amino acid 276, has previously been associated with risks of pyelonephritis and vesicoureteral reflux in childhood a priori hypothesis of their potential association with a cellular function. Even if a strict Bonferroni correction were taken for the association of TLR2_G2258A with ASB, the adjusted P value would remain significant at <0.017 (17 polymorphisms x <0.001 for high vs no ASB comparison). Regardless of which adjustments are chosen for our data, convincing evidence of a genetic effect ultimately requires replication studies as well as detailed analysis of functional effects of each polymorphism. Another limitation is that our cross-sectional study design does not enable us to assess whether the ASB episodes in our population are linked to a future occurrence of cystitis. We recently completed a prospective study of women with recurrent cystitis that included daily urine sampling prior to an episode of cystitis Potential limitations of our study include effects of population admixture and multiple comparisons. To account for possible confounding effects from population heterogeneity, we performed our genetic analyses in the Caucasian subgroup as well as the entire cohort. Despite these attempts to minimize population admixture effects, we cannot exclude this possibility. A second possible limitation is the issue of multiple comparisons. As a matter of hypothesis testing, we prioritized seven well-characterized TLR pathway functional polymorphisms and 10 SNPs in the two major receptors involved in neutrophil chemotaxis. SNPs with well-characterized function do not generally have the same requirement for adjustments for multiple comparisons, due to a well-founded Our study also has numerous strengths. First, to our knowledge this is the largest study to examine associations of gene polymorphisms and susceptibility to ASB, and is the only study to date to examine this phenotype in adults. Second, we used a population-based sampling strategy which increases the likelihood that our findings are representative of the population. Finally, this is the first study to examine the association of polymorphisms with urine chemokine and neutrophil levels.In summary, our results suggest that genetic variation of TLR and chemokine pathways is associated with asymptomatic bacteriuria and urine chemokine levels. Examining genetic regulation of the urinary tract immune response during ASB provides an opportunity to understand early events in pathogenesis prior to the complex cytokine and chemokine inflammatory cascades present during symptomatic cystitis episodes.Table S1CXCR1, CXCR2, & TLR polymorphisms & ASB (Caucasian only).(0.22 MB RTF)Click here for additional data file.Table S2CXCR1, CXCR2, & TLR polymorphisms & ASB secondary to gram-negative uropathogens for Caucasian only.(0.18 MB RTF)Click here for additional data file.Table S3CXCR1, CXCR2, & TLR polymorphisms & ASB secondary to gram-positive uropathogens for Caucasian only.(0.17 MB RTF)Click here for additional data file.Table S4CXCR1 and CXCR2 polymorphisms & clinical history of rUTI or pyelonephritis.(0.11 MB RTF)Click here for additional data file.Table S5CXCR1 and CXCR2 polymorphisms & clinical history of rUTI or pyelonephritis for Caucasians only.(0.12 MB RTF)Click here for additional data file.


There are conflicting reports on whether iron deficiency changes susceptibility to seizures. We examined the hypothesis that iron deficiency is associated with an increased risk of acute seizures in children in a malaria endemic area.We recruited 133 children, aged 3–156 months, who presented to a district hospital on the Kenyan coast with acute seizures and frequency-matched these to children of similar ages but without seizures. We defined iron deficiency according to the presence of malarial infection and evidence of inflammation. In patients with malaria, we defined iron deficiency as plasma ferritin<30µg/ml if plasma C-reactive protein (CRP) was<50mg/ml or ferritin<273µg/ml if CRP≥50mg/ml, and in those without malaria, as ferritin<12µg/ml if CRP<10mg/ml or ferritin<30µg/ml if CRP≥10mg/ml. In addition, we performed a meta-analysis of case-control studies published in English between January 1966 and December 2009 and available through PUBMED that have examined the relationship between iron deficiency and febrile seizures in children.p = 0.230) nor status epilepticus and it did not affect seizure semiology. Similar results were obtained when children with malaria, known to cause acute symptomatic seizures in addition to febrile seizures were excluded. However, in a meta-analysis that combined all eight case-control studies that have examined the association between iron deficiency and acute/febrile seizures to-date, iron deficiency, described in 310/1,018(30.5%) cases and in 230/1,049(21.9%) controls, was associated with a significantly increased risk of seizures, weighted OR 1.79(95%CI 1.03–3.09).In our Kenyan case control study, cases and controls were similar, except more cases reported past seizures. Malaria was associated with two-thirds of all seizures. Eighty one (30.5%) children had iron deficiency. Iron deficiency was neither associated with an increased risk of acute seizures (45/133[33.8%] cases were iron deficient compared to 36/133[27.1%] controls, Iron deficiency is not associated with an increased risk of all acute seizures in children but of febrile seizures. Further studies should examine mechanisms involved and the implications for public health. Plasmodium falciparum malaria has been described as the leading cause in children 6 months and older Acute seizures are common in children admitted to district hospitals in sub-Saharan Africa over 10% of admissions may report seizures. Infections are the predominant precipitants and in malaria endemic areas, Iron deficiency is also common in the same regions of Africa. Although the results are conflicting, some reports from other settings suggest an increased prevalence of febrile seizures in iron deficient children Iron is involved in several brain processes such as neuro-metabolism, myelination, and, neuro-transmitter function. The metabolism of several neuro-transmitters and monoamine and aldehyde oxidases is reduced in patients with iron deficiency  and mean cell volume (MCV), serum iron, ferritin, transferrin saturation and soluble transferrin receptor levels (sTfR) and/or bone marrow aspirate staining are used. Levels of most of the biochemical markers are altered during an acute infection and may therefore be unreliable. Although sTfR is a sensitive indicator of iron status in adults The diagnosis of iron deficiency is problematic in children with acute febrile illnesses and even more so, in those with As part of an epidemiological study examining infectious, biochemical and genetic risk factors for acute seizures in rural Africa This was a case control study of children with acute seizures (cases), or age-matched controls without seizures, admitted to Kilifi District hospital, on the coast of Kenya.Ethical approval for the study was granted by the Kenya Medical Research Institute and a written consent was obtained from parents or guardians of each participating child.Kilifi District Hospital serves a predominantly rural population and is the only hospital in the area where the majority of very sick children are admitted for treatment. Annually, it admits about 5,000 children under the age of 14 years. A demographic surveillance system that defines an area where the majority of patients attending the hospital live has been in place since 2001. This study area undergoes regular census three times a year. Each person has a unique identifier that individually links the census data to hospital admissions. Common diagnoses include pneumonia, malaria, severe malnutrition, diarrhea, and neonatal diseases. Lead poisoning is not a recognized problem in this rural area that has no industrial establishments.Plasmodium falciparum malaria which is responsible for up to 2/3 of all acute seizures in children in the region, may be acute symptomatic rather than febrile seizures and making this distinction is difficult We estimated that at 80% power and 5% level of significance, with a ratio of cases to controls of 1∶1, a matched sample size of 132 cases and 132 controls could detect an odds ratio of 2.2. Cases were children from the surveillance area, aged 3–156 months, admitted to Kilifi District hospital with acute seizures during the presenting illness. Acute seizures were defined as reported or observed repeated rhythmic and involuntary muscle contractions or jerky movements of the limbs, face or mouth that was not stimulus sensitive. We chose to recruit all children with all acute seizures The next child to be admitted, of similar age to the case, from within the study area, with any diagnosis but no seizures, was recruited as a control to form the comparison group. Age matching within epochs of 3–12, 13–24, 25–36, 37–48, 49–60 and >60 months. Controls that developed seizures during hospitalization were renamed cases and another control recruited. Children with epilepsy (defined as 2 lifetime episodes of unprovoked seizures) and significant developmental delay were excluded.In Kilifi hospital, all children have standardized clinical and laboratory data prospectively collected on admission and at discharge or death and directly entered into a computer database. As part of a bigger epidemiological study on acute seizures, we modified this database to obtain additional data relevant to acute seizures On admission, the admitting clinician, usually a Medical or Clinical Officer, performed resuscitation and emergency care procedures such as correction of hypoxemia, hypoglycemia and hypovolemia for all cases and controls using the same standard guidelines After stabilization, children with iron deficiency were offered ferrous sulphate 20mg/kg/day for 30 days, and mebendazole, 100mg twice daily for 3 days. No follow up was however performed to determine whether this intervention was successful. The discharging clinician made the final diagnosis after review of the admission history, inpatient management notes and laboratory investigations. These diagnoses were checked by a supervising clinician and followed the current World Health Organization (WHO) guidelines for the management of common illnesses in hospitals with limited resources Venous blood was obtained for a full count , glucose , blood smears for malaria parasites (Giemsa stained) and microbiological culture. Plasma from heparinized tubes was separated by centrifugation. Part of the plasma was used to determine levels of electrolytes and creatinine and the remaining immediately frozen together with the cell pellet and stored at −80°C. After completion of the study, plasma ferritin, iron and transferrin were assayed using Vitalab Selectra E, Clinical Chemistry Analyser . DNA was extracted from the cell pellet using PUREGENE® DNA extraction kit  according to the manufacturers instructions and α-thalassemia genotyping for the common African 3.7kb α-globin deletion was performed employing PCR and gel electrophoresis methods described previously In children with malaria parasitemia, iron deficiency was defined as plasma ferritin<30µg/ml if CRP<50mg/ml or as ferritin<273µg/ml if CRP≥50mg/ml. In those without malaria parasitemia, iron deficiency was defined as plasma ferritin<12µg/ml in the absence of an inflammatory process (CRP<10mg/ml) or as ferritin<30µg/ml if CRP≥10mg/ml. The criterion offers a sensitivity of 75% and a specificity of 76%Anemia was defined as a hemoglobin concentration of <11g/dl. Microcytosis was defined as MCV below the age-corrected normal values for erythrocyte volumes  Individual clinical data was directly entered in a FileMaker 5.5 database at admission. Data was analyzed using Stata version 9.2 . To examine the relationship between iron deficiency and acute seizures, we compared the proportions of cases and controls with iron deficiency using Pearson's chi square test. An analysis for trend was performed to examine a “dose effect” relationship between plasma ferritin level or proportion with iron deficiency and the number of seizures. In addition, the association between iron deficiency and the type and the proportion of patients with status epilepticus (effect on seizure duration) and outcome were assessed. Continuous measures that were approximately normally distributed data were compared using the unpaired student's t-test while markedly skewed data were compared using Wilcoxon's rank-sum test. A p-value of <0.05 was considered significant.To be included in the meta-analysis, studies had to fulfill the following criteria:Published in English between January 1966 and December 2009Available through PUBMEDOriginal study involving childrenOf case-control or comparative study design with clear comparative groups of cases with seizures and controls without seizures andA specified criteria defining iron deficiency in the study subjects. Where this information was inadequate, the details could be obtained from the authors.Using the search phrase “iron deficiency” and “seizures” or “convulsions” for studies in PUBMED published between January 1966 and December 2009, we identified 55 publications. These were screened for eligibility and only 9 were selected, full texts of which were obtained Data from the eight studies was extracted into a standardized excel form and the results summarized together with the meta-analysis. The study variables included the total number of cases and controls in each study and the numbers and proportions in each group defined as being iron deficient. In addition, we obtained information on the definition used for iron deficiency. However, because the majority of studies used different tests or combination of tests to define iron deficiency, we neither did nor could we standardize the definition for iron deficiency. In one study The meta-analysis was also performed using STATA version 9.2. The risk ratios of the original studies are reported together with the combined estimate and 95% confidence intervals.One hundred and thirty three children admitted to Kilifi District Hospital between January 2005 and January 2006 with acute seizures, were age-matched to the same number of hospital controls. The mean age, nutritional status and gender of cases and controls, were similar . Childrep = 0.230, the mean and median levels of the biochemical markers of iron metabolism and the proportions of patients with iron deficiency and iron deficiency anemia were similar in cases and controls, A total of 81 (30.5%) children were iron deficient. Neither iron deficiency nor iron deficiency anemia was associated with acute seizures; 45/133 (33.8%) cases were iron deficient compared to 36/133 (27.1%) controls, Pearson's chi square 1.4378, We examined for a dose – response relationship between plasma ferritin or iron deficiency and the number of seizures the patient had during the illness. In either case, no dose response effect was observed . In addiBecause malaria was over represented in cases in comparison to controls , using a differential definition for iron deficiency as we did may introduce bias where the malaria specific definition is used more often in cases than in controls. We therefore performed a second analysis using a single definition for iron deficiency that disregarded the effects of malaria on ferritin levels but only stratified the ferritin levels by the inflammatory response, i.e. plasma ferritin<12µg/ml if CRP<10mg/ml or ferritin<30µg/ml if CRP≥10mg/ml. Such a definition led to an under ascertainment identifying only 43 children as iron deficient, yet, still, no association was observed between iron deficiency and acute seizures. Thus, only 20 (15%) cases had iron deficiency using the revised definition compared to 23 (17.3%) controls, Pearson's Chi Square 0.2497, p = 0.617.Plasmodium falciparum malaria is epileptogenic and some of the seizures in this infection may be acute symptomatic seizures rather than febrile seizures we performed a second analysis excluding all patients with malaria. Although the sample was small, even then, the proportions of patients with iron deficiency among cases and controls were similar; 13/44 (29.6%) of the remaining cases had iron deficiency compared to 22/91 (24.2%) of controls, p = 0.505.All previous studies included only children thought to have febrile seizures. Because We performed a conditional regression analysis to exclude possible confounding effects by nutritional status (weight for age), past seizures, fever and malaria, on the association between iron deficiency and acute seizures. Again, we found no independent association between iron deficiency and acute seizures. Instead, past seizures , fever , and malaria  were independently associated with acute seizures.To examine the effect of iron deficiency on seizure manifestation/semiology, we compared the type , number (including recurrences after admission) and duration (proportion with status epilepticus) of seizures in cases with and without iron deficiency. There was no association between iron deficiency and seizure manifestation or recurrences in the ward. In addition, iron deficiency was not associated with an increased risk of status epilepticus. All children who presented with impaired consciousness or coma and all three who died were non iron-deficient, All eight studies were hospital based and other than our study, only included children in the febrile seizure age group, 3 months – 6 years. However, each study used a different definition for iron deficiency  with dif2>50% and moment-based estimate of between studies variance = 0.469, A total of 1,018 children with seizures and 1,049 without seizures were included in the analysis. Overall, iron deficiency was associated with an increased risk of seizures: iron deficiency was reported in 310(30.5%) cases and 230(21.9%) controls, weighted OR (random effects model) 1.79 (95% CI 1.03–3.09), Test for heterogeneity: Q = 37.397 on 7 degrees of freedom (p<0.001), IThis study set out to examine the hypothesis that iron deficiency is associated with an increased risk of acute seizures in children. In those with seizures we further examined whether iron deficiency influences seizure manifestation, number or duration. We found no association between iron deficiency and acute seizures, and among cases, iron deficiency did not influence the manifestation or the number of acute seizures, the proportion with status epilepticus nor further recurrences in the ward. However, a meta-analysis of all eight case-control studies that have examined the relationship between febrile seizures or acute seizures and iron deficiency to-date suggested that iron deficiency may be associated with an increased risk of febrile seizures in children.The relationship between iron deficiency and febrile seizures has been controversial. Most of the early and conflicting studies i) examined the relationship between iron deficiency and febrile seizures; ii) had small sample sizes and iii) used different markers and definitions for iron deficiency and iv) lead toxicity and celiac disease were also raised as possible confounding factors A closer look at the studies which did not demonstrate an increase in seizures with iron deficiency reveals possible mixed phenotypes: other than the Iranian study Overall, these findings appear to demonstrate that iron deficiency is associated with an increased risk of febrile seizures specifically but not all acute seizures. Iron deficiency may be an important factor in the pathogenesis of febrile seizures but not all acute seizures in children with febrile illnesses. The use of different criteria for the diagnosis of iron deficiency is clearly a weakness and calls into question the use of a combined estimate in the meta-analysis. Despite this weakness, the pattern of results suggests a clear pattern of association between iron deficiency and febrile seizures but not necessarily so with all acute seizures. Neuro-developmental impairments secondary to iron deficiency may provide a background of abnormal neuron functioning and increase the risk of seizures with subsequent provoking events. Other factors e.g. the aetiological cause may be more important in acute symptomatic seizures.As shown in an animal model, other possible mechanisms may include altered neuron excitability and neurotransmission p = 0.074 but a reduction in seizure recurrences during admission Findings from the iron supplementation study in Tanzania that was terminated early because of increased all cause mortality and admissions to hospital First, the population in this study was different from previous studies in that it was not limited to children with febrile seizures but included all children admitted with acute seizures associated with malaria which may not be febrile seizures Third, the use of only hospital controls in this study may have introduced a selection bias since these patients are more likely to have higher levels of iron deficiency than does the reference population. A better design would have included two sets of controls: hospital and community controls. Our study is however comparable to all the previous 7 case control studies that have examined the association between iron deficiency and seizures. All have only used hospital controls as the only comparative group. In this regard, we are comparing like to like. We also took additional measures to reduce such bias in our inclusion criteria namely, all controls came from the same geographical area as the cases  and were matched for age. More importantly, in an earlier survey of the same geographic area that aimed to establish hematological indices in children in the community and which defined iron deficiency using the same parameters as in the present case control study, we had established that 116/311 (37.3%) well children in this community were iron deficient, . A second study in the same area had documented similar results earlier Other possible bias includes recall and information bias  and misclassification error resulting from definitions used for classifying iron deficiency. Fourth, unlike all the other studies included in the meta-analysis, subjects in our case control study had a very wide age range. Other than young infants (0–2 months old), we included all patients in the paediatric age group admitted to our children's wards (up to 13 years of age). It would have been more appropriate if only children 3months - 6 years were included. This not only makes comparison across studies difficult but also the aetiology of acute seizures may be different In conclusion, although our study found no association between iron deficiency and all acute seizures, an analysis of the individual studies and the combined result of case control studies to date suggest that iron deficiency is associated with an increased risk of febrile seizures in children. Further studies should examine the mechanisms involved, the possible association with subsequent epilepsy after severe malaria, and the implications for public health programs.


Observational studies, including the case-control design frequently used in epidemiology, are subject to a number of biases and possible confounding factors. Failure to adjust with them may lead to an erroneous conclusion about the existence of a causal relationship between exposure and disease. The Cochran–Mantel–Haenszel (CMH) test is widely used to measure the strength of the association between an exposure and disease or response, after stratifying on the observed covariates. Thus, observed confounders are accounted for in the analysis. In practice, there may be causal variables that are unknown or difficult to obtain. Hence, they are not incorporated into the analysis. Sensitivity analysis enables investigators to assess the robustness of the findings. A method for assessing the sensitivity of the CMH test to an omitted confounder is presented here. The technique is illustrated by re-examining two datasets: one concerns the effect of maternal hypertension as a risk factor for low birth weight infants and the other focuses on the risk of allopurinol on having a rash. The computer code performing the sensitivity analysis is provided in appendix A. Observational studies are commonly used to evaluate the effect of treatments or chemical exposure, e.g. the association between risk factors and a subsequent event. Unlike randomized experiments, researchers cannot control the assignment of treatments to subjects to ensure that subjects receiving different treatments or exposure are similar with respect to major covariates. The Cochran–Mantel–Haenszel CMH;  test is U, and treatment assignment, and then recomputes the test statistics and p values for a range of plausible differences in the prevalences of the confounder in the two groups. If a moderate degree of confounding could change the inference about the treatment effect on the outcome, then one questions the soundness of the conclusions. U and treatment assignment and that between U and response are jointly modelled. This simultaneous form allows one to incorporate subject matter knowledge about both relationships into the sensitivity analysis.Since observational studies remain subject to unobserved confounding, one should explore how sensitive the results are to a possible omitted variable. Extensive work has been done on how to perform sensitivity analysis for observational data by This paper extends the ‘simultaneous’ approach to examine the sensitivity of the CMH test to an unobserved confounder. The rest of this paper is organized as follows. X be the exposure, e.g. environmental hazard, genetic factor, radiation exposure; and Y be the outcome, e.g. disease, mortality, or in an employment discrimination case, being promoted or passing a pre-employment exam. We are interested in testing the causal relationship between X and Y using the CMH test to control for other known factors related to the response. Usually the data are organized into J 2×2 tables, where in each stratum the covariates have similar values. The data for the jth stratum  are shown belowLet πj1 (πj0), j=1, …, J, be the probability that subjects who are exposed (not exposed) in stratum j has an event. In terms of these parameters, the odds ratio between exposure and outcome in stratum j isθ. Let J 2×2 tables. When the responses for each group in all strata follow independent binomial distributions, the full data have the product binomial likelihood functionLet H0: πj0=πj1, j=1, …, J or H0: θ=1. H0: θ=1 versus H1: θ>1 iswj=mj1mj0/Nj, πj1 and πj0, respectively. Using the conditional central hypergeometric likelihood rather than the product binomial If all the confounders are observed and controlled by stratification, then the null hypothesis of no-exposure effect is θ=1 ; the test statistic and the p value are calculated using p values with respect to different parameters for confounding can be used to assess the plausibility that the current inference could be altered due to the effect of an unobserved factor.If the strength parameter  β=0 see . Then thC=exp (γ) measures the effect of U on binary response Y and D=exp (δ) measures the increasing odds of U being positive when X increases by one unit.The proposed sensitivity analysis will be applied to data from two observational studies. In these applications, X) is maternal hypertension and the event of interest is LBW infant (Y).Data on 500 singleton births in a London Hospital  were usep=0.015 and the odds ratio is 2.74 with 95% CI . The Breslow–Day test for homogeneity shows that the odds ratio estimates are homogeneous across different strata (p=0.785). Is it safe to draw the conclusion that maternal hypertension is causally related to LBW?‘Advanced’ maternal age is defined as any expectant mother who will have reached her 35th birthday by delivery date. The mothers are divided into ‘young’ and ‘advanced’ age group according to this criterion. The number of infants with normal birth weight (NBW) and LBW by sex, gestation status, maternal age and maternal hypertension are shown in http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5127a1.htm). Hence, race is an important risk factor with an apparent odds ratio of approximately 2.0–2.4 for LBW (U) because it is a recognized risk factor. We assume that the mother's race is not related to the sex of their babies, but that the percentage of mothers who are black varies across the maternal age groups as well as gestation term. The possible scenarios for the percentage of black mothers by maternal age and gestation term are shown in Despite substantial reductions in US infant mortality during the past several decades, black–white disparities in infant mortality rates persist. Important determinants of racial/ethnic differences in infant mortality are LBW, defined as less than 2500 g . The num for LBW . A recenp values of the adjusted CMH test with respect to different values of C and D for scenarios 1–5. Note that the strength parameter C=exp (γ) is the odds ratio of a black mother having a LBW baby compared with a white mother. The imbalance parameter D=exp (γ) is the increased odds ratio of a mother being black if she has hypertension. When C=1 or D=1, race is not a confounder and the CMH test is unchanged.C and D, which increase the p value of the CMH test to 0.05. C and D, which corresponds to p=0.05. From C=1.5 and D=1.1, then the two-sided p value would reach the 0.05 level. Thus, both p value of the CMH test is sensitive to moderate changes in C and D, indicating that the significant association between maternal hypertension and LBW might have arisen from a hidden bias due to confounding. Hence, other studies, including race and ethnicity information, are needed to reach the conclusion that maternal hypertension causes LBW. If the studies demonstrate that increased incidence of black mothers with hypertension was slight with odds ratio less than 1.5, then the significance of the London study would not be affected by the omission of race.Another way to perform the sensitivity analysis is to obtain the threshold values of the sensitivity parameters Although in Rubin's causal model, sex and race are not used as ‘treatment’ variables in causal inference because it is difficult to ascribe causality to variables that cannot be altered , here rap<0.0001. In a randomized trial or in the absence of unobserved confounders, this indicates strong evidence that allopurinol causes rash  and no amoxicillin (0). Again, the prevalences of using amoxicillin among the non-allopurinol users are denoted by qj, j=1, …, J. The parameter δ indicates the imbalance of U between allopurinol users and non-users. The parameter γ indicates the increasing probability of having rash among users of amoxicillin. We assume two possible scenarios for the prevalences of amoxicillin use among the non-allopurinol users, i.e. qj=10% or 20%, where j=1, 2.Although information about the other drug use and users' allergic history was not collected, one can assess the sensitivity of the CMH test with respect to an unobserved confounder. Let C=4 and the imbalance parameter D=4, the CMH test remains highly significant with p=0.002. This example also illustrates the additional information provided by the simultaneous sensitivity analysis compared with the earlier approaches by Γ, which is similar to D in our analysis, to measure the imbalance of treatment assignment with respect to the unobserved factor. The upper bounds on the p values based on his analysis are 0.036 and 0.30, for Γ=2 and 3, respectively, which indicates that the causal relationship between allopurinol use and rash could have arisen from an unobserved confounder that triples the risk of rash. While such a confounding is unlikely to exist, the conclusion based on the simultaneous analysis is stronger. Even when C=∞, the test barely reached non-significance for D=3 and qj=10%. This sensitivity analysis is more stringent, indicating the conclusion that allopurinol causes rash is very robust to an unobserved confounder. This is consistent with the current medical practice that lists rash as the one common side effect of allopurinol use (http://www.nlm.nih.gov/medlineplus/druginfo/medmaster/a682673.html).The increasing odds ratio of having rash for amoxicillin users compared with non-users is not known, but it is likely to be below 4. Based on plausibly be explained by an unobserved confounder before drawing a firm conclusion.Because the CMH test is widely used to analyse the data obtained in epidemiological studies, legal cases and social sciences, it is important to conduct a sensitivity analysis to answer whether that inference could γ and δ (The proposed simultaneous sensitivity analysis approach is similar to the primal sensitivity analysis method by At the design stage, it is also useful to examine the size and power of the corrected CMH test in the presence of omitted confounders in a range of realistic values of the strength and imbalance parameters. The calculation of size and power for the Cochran–Armitage trend test has been derived by In future studies, we plan to extend this methodology of sensitivity analysis to data where the unobserved confounders are continuous and ordinal. In real-world observational studies, there may be more than one omitted confounder; background information on the joint distribution of the multiple confounders can then be used to replace the univariate distribution


Plasmodium falciparum malaria.A SNP at position 131, in the FcγRIIa gene, affects the binding of the different IgG subclasses and may influence the clinical variation seen in patients with falciparum malaria. This study confirms and extends previous findings, analysing the FcγRIIa (CD32) polymorphism in relation to the IgG subclass distribution seen among two sympatric tribes living in eastern Sudan, characterized by marked differences in susceptibility to P. falciparum malaria infection were genotyped for the FcγRIIa-131 polymorphism. For comparison, 101 non-Fulani donors –  – living in the same study area, were genotyped. The levels of plasma antibodies (IgG and subclasses) to four malaria antigens  were measured using indirect enzyme-linked immunosorbent assays.Two hundred and fifty Fulani subjects living in an area of meso-endemic The FcγRIIa-H/H131 genotype was found to be significantly more prevalent in the Fulani as compared to the non-Fulani ethnic groups . The Fulani showed lower anti-malarial IgG1 and IgG3 antibody levels as compared to the non-Fulani and higher levels of IgG2 antibodies.The FcγRIIa-H/H131 genotype and H131 allele is at higher frequency in the Fulani ethnic group. The H/H131 genotype was consistently associated with higher levels of anti-malarial IgG2 and IgG3 antibodies, while the R/R131 genotype was associated with higher levels of IgG1 antibodies. Plasmodium falciparum malaria  with 95% confidence intervals [CI] that did not cross 1.00 with P value < 0.05, defined as statistically significant. The FcγRIIa-His/Arg131 group was used as a reference in the analyses, because this genotype is the most prevalent in human populations . An overA total of 351 individuals were included in the study, 202 (63.2%) females and 129 (36.8) males, with an overall age range of 2 to 75 years. No donor had any malaria parasites detectable by microscopy at the time of sampling; this result was confirmed by PCR screening of the samples (not listed).All individuals of the two study groups were genotyped for the FcγRIIa-H/R131 SNP. The H/H131 genotype was at higher frequency in the Fulani than in the non-Fulani ethnic group   , and also tended to have higher IgG3 antibody levels (P = 0.062) , IgG3 (P = 0.01) and IgG4 antibodies against AMA-1 (P = 0.01) Table . No suchRegarding antibodies reactive with MSP2-3D7 or -FC27 in the study population as a whole, a similar pattern as with AMA-1 reactive antibodies was seen. The H/H 131 genotype carriers showed statistically significantly higher levels of IgG2 , IgG3 (P = 0.01 for both antigens) and IgG4 , while the R/R 131 genotype carriers showed higher levels of IgG1 antibodies (P = 0.01 for both antigens)  and IgG3 , while Fulani H/H 131 carriers showed higher levels of IgG2  and IgG4 antibodies   and IgG3 (P < 0.001) Table . FurtherInterethnic comparison of Pf332-C231 reactive antibodies in relation to FcγRIIa genotypes showed that non-Fulani H/H 131 carriers had significantly higher levels of IgG2  and IgG4 , IgG3 antibodies were at significantly higher levels in H/H 131 carriers of the Fulani group  (Table P. falciparum parasite densities and parasite rates than the neighbouring ethnic group, the Masaleit [The present study confirms and extends the previous finding in the same study population, that the prevalence of the FcγRIIa-H/H131 genotype and the H131 allele are statistically significantly higher in Fulani than in sympatric non-Fulani children . The preMasaleit . WhetherP. falciparum infection, compared to the R/H131 heterozygotes [Contrasting results have been reported regarding associations of FcγRIIa-R/H131 genotypes with malaria disease outcomes . Thus, oozygotes . Anotherozygotes -31. The ozygotes , while iozygotes . The disSeveral studies in Burkina Faso and Mali have shown that the Fulani have significantly higher levels of anti-malarial antibodies than other sympatric ethnic groups ,10,13, iWhile the FcγRIIa-R131 molecule shows a similar binding of the cytophilic subclasses IgG1 and IgG3 ,36, the There is accumulating evidence for IgG1 and IgG3 antibodies playing an important role in protection against malarial disease . HoweverThe present study confirms and extends the previous findings that the FcγRIIa-H/H131 genotype and the H131 allele are more frequent in the Fulani, than in their sympatric ethnic neighbours. The Fulani in Sudan had lower anti-malarial IgG1 and IgG3 antibody levels than the non-Fulani and higher levels of IgG2 antibodies. The higher IgG2 antibody levels seen in the Fulani may be due to the relatively high prevalence for the H/H131 genotype in this group. Further studies are needed to elucidate if the FcγRIIa-R/H131 polymorphism may be a contributing factor to the differential susceptibility to malaria seen in different ethnic groups.The authors declare that they have no competing interest.AN carried out the sampling, the genotyping of FcγRIIa polymorphism and participated in the statistical analysis. NCI performed the ELISA and also participated in the statistical analysis. Both AN and NCI drafted the manuscript. GE, KB, HG and MT designed the study and set up the framework, financed and revised the manuscript. HAB and RFA prepared and provided the antigens used in the study and participated in the manuscript preparation. All authors read and approved the final version of the manuscript.


Season of birth (SOB) has been associated with many physiological and psychological traits including novelty seeking and sensation seeking. Similar traits have been associated with genetic polymorphisms in the dopamine system. SOB and dopamine receptor genetic polymorphisms may independently and interactively influence similar behaviors through their common effects on the dopaminergic system.DRD2) TaqI A and D4 (DRD4) 48 bp VNTR. Primary analyses included factorial gender×SOB ANOVAs or binary logistic regression models for each dependent trait. Secondary analysis extended the factorial models by also including DRD2 and DRD4 genotypes as independent variables. Winter-born males were more sensation seeking than non-winter born males. In factorial models including both genotype and season of birth as variables, two previously unobserved effects were discovered: (1) a SOB×DRD4 interaction effect on venturesomeness and (2) a DRD2×DRD4 interaction effect on sensation seeking.Based on a sample of 195 subjects, we examined whether SOB was associated with impulsivity, sensation seeking and reproductive behaviors. Additionally we examined potential interactions of dopamine receptor genes with SOB for the same set of traits. Phenotypes were evaluated using the Sociosexual Orientation Inventory, the Barratt Impulsivity Scale, the Eysenck Impulsivity Questionnaire, the Sensation Seeking Scale, and the Delay Discounting Task. Subjects were also asked about their age at first sex as well as their desired age at the birth of their first child. The dopamine gene polymorphisms examined were Dopamine Receptor D2 (These results are consistent with past findings that SOB is related to sensation seeking. Additionally, these results provide tentative support for the hypothesis that SOB modifies the behavioral expression of dopaminergic genetic polymorphism. These findings suggest that SOB should be included in future studies of risky behaviors and behavioral genetic studies of the dopamine system. In the retina DRD4 mRNA has been found in photoreceptor cells that indirectly control melatonin synthesis and have a regulatory role on light sensitive cyclic adenosine monophosphate Consistent with the dopamine-melatonin hypothesis, traits associated with SOB have also been linked to genetic polymorphisms in the dopamine system. These include BMI This paper focuses on SOB and its interaction with dopamine receptor genes and gender. It is part of a larger series of studies examining the associations of genetic polymorphisms of the dopamine system with behavior DRD2 TaqI A and DRD4 48 bp VNTR. The DRD2 TaqI A site is a single nucleotide polymorphism (SNP) with a major A2 allele, and minor A1 allele. The A1+ genotype (heterozygous or homozygous A1) has been most strongly associated with substance abuse, particularly alcoholism, albeit with some controversy DRD2 TaqI A site is 9.4 kb downstream from the coding region for the dopamine D2 receptor gene. It is not in any known regulatory region, and although the A1 allele is associated with a decrease in dopamine D2 binding and glucose metabolic rates in many brain regions DRD2 expression is unknown. The TaqI A polymorphism is also in a nearby kinase gene, the Ankyrin Repeat and Kinase Domain Containing 1 (ANKK1) gene, where it causes a Glutamate→Lysine substitution TaqI A that could easily account for these associations We examined two dopaminergic genetic polymorphisms: DRD4 48-bp VNTR polymorphism is in exon 3 of the gene coding for the dopamine receptor D4. The VNTR polymorphism varies between 2 and 11 repeats of a similar 48-bp coding region sequence, with a trimodal distribution of 2, 4 and 7 repeat alleles  in most, but not all, populations DRD4 VNTR polymorphism has not been definitively characterized, long alleles  have been generally found to be functionally less reactive in in-vitro expression experiments The Based on the existing literature, we predicted that winter-borns would exhibit increased sensation seeking, increased impulsivity, more promiscuous sexual behavior and a desire for children earlier. We further predicted that being winter-born would potentiate the effects of risk-conferring dopamine receptor alleles on these behavioral traits.Between February and April 2005 a total of 195 subjects were recruited for participation from the Human Subject Research Pool at the State University of New York at Binghamton, U.S.A. The subject pool draws on mostly full time students currently in psychology courses who participate in research studies for course credit. No screening measures were placed on who could participate. All procedures were approved by the Human Subjects Research Review Committee at the State University of New York at Binghamton and all subjects gave informed consent. Participants attended group sessions (maximum = 10), where they were first provided with oral instructions followed by DNA sample collection. Participants then completed the delay discounting task followed by the self-report measures administered in random order. In addition to the oral instructions, the DDT task and other measures were accompanied by on-screen written instructions and experimenters were available throughout the sessions for questions. The sessions lasted approximately one hour. All data were collected via personal computers.The SOI measures restriction of sexual and pair-bonding behaviors. It has been validated on student populations around the world The BIS-11 provides a general measure of impulsivity and three subscores: Attentional Impulsiveness (BIS-AI), Motor Impulsiveness (BIS-MI) and Non-Planning Impulsiveness (BIS-NPI). The BIS-11 has undergone psychometric validation The EIQ is a self-report measure of impulsivity that generates three subscales, of which two were relevant to the current study: Impulsiveness (EIQ-I), and Venturesomeness Sensation seeking is a related construct to impulsivity, and has both been shown to exhibit moderate positive correlations with self-reported impulsivity and to potentially share genetically-mediated common biological mechanisms with impulsivity k). The DDT was administered with hypothetical money via a custom computer program k values are calculated as 2R values. Erratic subjects and those with 2R values below 0.30 were excluded from principal analyses k value was normalized with a logarithmic transformation, as is typical in delay discounting research.To capture discounting of delayed rewards empirically, the DDT poses participants with repeated choices between a smaller reward received immediately and a greater reward received after some time delay . Over the course of the task, the amounts of immediate rewards are successively modified, as is the duration of delay. The individual's responses to the entire array of choices are then used to empirically derive their discounting function ').DNA was collected with QuickExtract buccal swabs and extracted with BuccalAmp solution as directed by the manufacturer (Epicenter). Subjects were instructed to rinse their mouths out with water before swabbing. DRD2 TaqI A and DRD4 VNTR genotypes has been conducted previously and are not reported on here DRD2 HW equilibrium was tested with Fisher's Exact and DRD4 with the Markov Chain algorithm.All data were examined for outlying data points, distribution normality, and missing values. To assure missing responses were not systematically biased by SOB, missing versus non-missing data for each phenotype scale was analyzed by SOB in 2×2 contingency tables. Examination of bias in missing data by DRD2 TaqI A polymorphism, individuals with at least one A1 allele were designated as A1+ and those who were homozygous for the A2 allele were designated A1-. Similarly, DRD4 48 bp VNTR genotypes were separated into long allele (7 repeats or longer) present (L+) and long allele absent (L−) groups. The principal data analyses used a tiered analysis. First, since several past studies have found that SOB effects vary by gender, factorial 2 ×2 (Winter-Born/Not-Winter-Born) ANOVAs were conducted. Then to see if genotype interacted with SOB to effect the traits in question, DRD2 and DRD4 genotypes were added to each factorial model . Similarly, categorical dependent variables were first analyzed with forward conditional factorial binary logistic regression models with dichotomized SOB and gender and then secondarily include DRD2 and DRD4. Direct SOB effects and SOB interactions with genotypes are the main focus of the study. Since this study is only concerned with sexual dimorphism in so far as it moderates SOB or genotype associations, main effects of gender are not commented on. All scales used in this study were previously examined in relation to the genetic polymorphisms and gender Based on previous association studies of the Since this study employs multiple phenotypic scales, the potential exists for type I errors. For several reasons we have not employed a correction of our significance criteria for multiple testing DRD2 TaqI A and DRD4 48-bp VNTR in Supplementary DRD2, Fisher's Exact test p = 1.0; DRD4 VNTR, Markov Chain Algorithm p = 0.38). Descriptive statistics of each dependent variable are given in The sample is described demographically in DRD4 or DRD2 genotypes (not shown). Between 2.8% and 14.5% of values were missing across phenotype scales (179 of the 195 subjects (91.8%) met the inclusion criteria for the SOB analysis. Winter borns made up 52.5% of the sample. SOB did not systematically vary with gender, e scales . No heteDRD2 and DRD4 were added as independent variables in the factorial ANOVA and binary logistic regression models. We found a significant interaction effect of DRD2×DRD4 on total sensation seeking , which was not observed in previous models that did not include SOB. Among those without A1 alleles (A1−), long DRD4 alleles (L+) were associated with decreased SSS-Total, but the reverse was true among those with A1 alleles (A1+). In addition, there was an interaction effect of SOB×DRD4 on EIQ-V  as illustrated in To evaluate whether there was a moderating relationship between SOB and dopamine gene polymorphisms on the behavioral traits in this study, DRD2×DRD4 analysis (not shown).All significant findings in the gender by SOB analysis (without genotype variables), were found in this gender×SOB×DRD4) seemed to be moderated by SOB.We hypothesized that winter-borns would show higher rates of risk-associated behaviors, including sensation seeking, impulsivity, and sexual promiscuity. We found support for this hypothesis only for sensation seeking and in a near-significant trend for virginity status. Additionally, we hypothesized that both winter-birth and risk-conferring alleles would together promote more risky-behaviors. We found only limited support for such an interaction. However, we did find that including SOB revealed some previously unobserved effects of the DA receptor genetic polymorphisms. On the EIQ Venturesomeness scale, the expression of a dopamine gene polymorphism  and potentially virginity  but not other related scales such as the Eysenck Impulsivity Questionnaire (EIQ) or Barrett Impulsivity Scale (BIS) which have been associated with the dopamine system While we hypothesized that SOB effects on behavior are related to dopamine-melatonin changes, with our dataset it is not possible to clearly distinguish the underlying basis for the behavioral effects of SOB. While the SSS has been related to the DA system, it has also been related to other neurotransmitters, enzymes and hormones DRD4 in predicting venturesomeness (EIQ-V) is consistent with our hypothesis that dopamine D4 receptors may be particularly mechanistically entangled with SOB effects. Such an interaction may indicate that both SOB and DRD4 act through related dopaminergic substrates. Additionally, the DRD2 by DRD4 association revealed when SOB was included in models together with DRD2 and DRD4 tentatively suggest that SOB and dopamine polymorphisms control overlapping traits. This DRD2×DRD4 associations with sensation seeking  is consistent with past findings of an interaction between DRD2 and DRD4 on impulsivity, where similarly the most extreme impulsive individuals were A1+/L+ Including SOB in genotypic models yielded two new significant findings not previously observed. One was an interaction effect between genotype and SOB. This interaction between SOB and The results presented here provide limited support for the hypothesis that SOB modulates expression of genetic polymorphisms of the dopamine system. Thus they add to previously published studies that have documented SOB interactions with the expression of dopamine system genetic polymorphisms, including interactive effects on psychiatric disorders Circadian rhythms have been found in virtually all organisms that have been studied This rapid evolutionary potential may also be innate in humans, and subject to natural selection. Although not readily apparent, SOB could be correlated with other factors that serve as reliable cues of the environmental quality an individual is likely to experience throughout their lifetime (e.g. infectious disease load or nutrition). In order for birth environment to serve as an adaptive cue to change behaviors, birth environment must be reliably correlated to the environment experienced many years later. Alternatively, SOB may cause adaptive behaviors in infants that are selectively advantageous enough to outweigh maladaptive behaviors later in life. While it is difficult to imagine how birth climate, would be a reliable signal of future environments beyond perhaps a few years One possible way such signals could be functional is if climate changes were a persistent survival problem for humans. There is evidence that “during the present (Holocene) interglacial…cold and dry phases…[occurred]… on a 1500-year cycle, and with climate transitions on a decade-to-century timescale” Alternatively, early photoperiod may have non-adaptive effects on development. Stressed pregnant rats have offspring with altered physiologies and behavior including altered dopamine levels DRD2 and DRD4 genetic polymorphisms. But, including SOB in factorial models revealed a previously unobserved association and evidence of moderation of DRD4 expression by SOB. These results must be viewed with caution as the number of phenotypic dependent variables analyzed here may have increased the risk of type I errors. An adaptive basis for the associations found here and in other studies of SOB is not clear. The behavioral implications of SOB remain ambiguous and its interactions with genotype effects are tentative. To further dissect the association of season of birth with later behaviors, more comprehensive analysis including experimental variations of photoperiod in lab animals in utero and early in life, and analysis of light exposure of pregnant women and later their young children should be conducted. However, SOB is an easy variable to collect (requiring only knowledge of the date and location of birth of subjects) that may help elucidate behavioral genetic associations. The current results along with those of past studies provide ample reason to include season of birth as at least a control in future studies of impulsivity/risky and sensation-seeking behaviors. Furthermore, season of birth should be included in future behavior genetic studies of the dopamine system.Our results tentatively suggest that SOB has a different, but related, psychological impact than dopamine D2/D4 receptor genetic polymorphisms on several behavioral phenotypes. This study replicates the past findings that winter-borns are more sensation seeking, but differs in that the association was only evident in males. SOB was unrelated to several behavioral/psychological phenotypes that were associated with File S1Delay Discounting Protocol(0.04 MB DOC)Click here for additional data file.File S2Datafile. Complete dataset from the study.(0.15 MB XLS)Click here for additional data file.File S3Genotype Protocols and Genotype and Allele Frequencies(0.07 MB DOC)Click here for additional data file.File S4Correlations Between Continuous Variables. Use Tabs to toggle between Female, Male and Combined Male and Female Correlations.(0.05 MB XLS)Click here for additional data file.


Idiopathic aortitis is a rare condition characterized by giant cell or lymphoplasmacytic inflammation of the aorta. The purpose of this study was to describe risk factors for the development of idiopathic aortitis.We conducted a case control study of 50 patients who were age-matched with two control subjects with non-inflammatory ascending aortic aneurysms. We examined whether the prevalences of gender, hypertension, hyperlipidemia, diabetes mellitus, smoking, family history of any aortic aneurysms, and elevated inflammatory markers differed between cases and controls.P = 0.014) and active smoking  to be associated with idiopathic aortitis. The association with smoking persisted after adjustment for gender . There was a trend toward lower prevalence of diabetes mellitus in cases  but no difference in prevalences of other risk factors. The median pre-operative erythrocyte sedimentation rate (ESR) was 20 mm/hour in cases (n = 13) and 9 mm/hour in controls (n = 22). The median pre-operative C-reactive protein (CRP) levels were 12 mg/L in cases (n = 8) and 3 mg/L in controls (n = 6) . A higher proportion of cases versus controls had elevations in ESR  and CRP .The mean age of cases was 71.6 ± 8.9 years and that of controls was 71.1 ± 8.9 years. We found female gender (odds ratio [OR] 2.41, 95% confidence interval [CI] 1.20 to 4.85; Gender and smoking may interact in complex mechanisms with immune and proteolytic pathways in older, less distensible thoracic aortas. Elevated acute-phase reactants as a marker of systemic inflammation may be present in some patients. Aneurysms of the thoracic aorta are rare and occur with an incidence of 5.9 per 100,000 . Rarely,The risk factors for development of aortic complications in GCA have been well studied . The preHistopathologic examination of the aortic wall revealed a paucity of inflammatory infiltrate but multiple foci of disruption of elastic lamellae, even in areas devoid of inflammation. There was increased expression of matrix metalloproteinase (MMP)-2 in the temporal artery as well as aortic tissue, whereas MMP-9 was found only in temporal artery specimens with active inflammation . Thus, tThe risk factors for development of idiopathic aortitis are not known. The problem is compounded by the fact that many patients are diagnosed post-operatively after histopathologic review of the surgical specimen reveals giant cell inflammation. Information on pre-operative traditional markers of inflammation such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) is scanty -9. In thMedical records of all patients at least 18 years old who underwent surgical resection for ascending aortic aneurysm from 1 January 2000 until 31 July 2006 were searched by means of a database of pathology specimens. Patients with giant cell or lymphoplasmacytic aortic inflammation were identified, and the histopathology slides were reviewed (DVM). Individuals with aortitis due to identifiable systemic rheumatic diseases, infectious diseases, and heritable diseases  were excluded from the analyses. Individuals who declined to have their medical records used for research were also excluded. Patients with idiopathic aortitis constituted our case group. The cohort of patients serving as controls was drawn from patients who were undergoing ascending aortic aneurysm repair during the same period and who fulfilled the exclusion criteria and did not have giant cell or lymphoplasmacytic inflammation in the wall of the resected aorta. For each case, two control subjects matched on age (± 5 years) and year of surgery were randomly selected from the pool of all controls with non-inflammatory non-infectious aneurysm.Age, gender, and race were abstracted from the patient medical records. Cardiovascular risk factors assessed included gender, presence of hypertension, diabetes mellitus (types I and II), hyperlipidemia, family history of aneurysms, and smoking. Presence of hypertension, hyperlipidemia, or diabetes mellitus at the time of surgery was identified in the medical record by ICD-9  coding or by physician diagnosis or the patient provided information on medical and family history during their visits to the Mayo Clinic . Family history of any aortic aneurysm was collected from clinical records or patient family history forms. Smoking history at the time of surgery was classified as never, current (within last 30 days), or former (quit more than 30 days ago). ESR and CRP values were recorded if they had been measured within a month pre-operatively.P values of less than 0.05 were used to denote statistical significance. For risk factors with a prevalence of 25% to 50%, the study had 80% power to detect an OR of 2.9. For risk factors with a lower prevalence  or a higher prevalence , this study had 80% power to detect an OR of 4.0. The study was approved by the Mayo Clinic Institutional Review Board (number 08-008786) and was conducted according to its guidelines.Descriptive statistics were used to summarize the data . The association between case/control status and cardiovascular risk factors was examined by means of logistic regression models. Each cardiovascular risk factor was examined individually and after adjustment for gender. Analyses are reported as odds ratio (OR) with corresponding 95% confidence intervals (CIs). The Fisher exact test was used to analyze percentage of cases versus controls with pre-operative elevation in ESR and CRP. In all cases, two-tailed P = 0.69).We identified 75 cases of non-infectious aortitis from patients who had undergone surgical repair during the study period. Of these, 25 cases were excluded, including patients with a history of GCA/PMR (n = 15), inflammatory arthritis (n = 2), Takayasu arteritis and Crohn disease (n = 1 each), bicuspid aortic valve (n = 3), and Marfan syndrome (n = 1). Two additional patients, one with a history of thymoma and one mislabeled as having aortitis without evidence of inflammation in the surgical specimen, were excluded. The clinical features, imaging findings, and surgical outcomes of 43 of these 50 patients with idiopathic aortitis have been described previously ,11. The P = 0.014). To reduce the probability of selection bias, we then compared the gender distribution of the 100 controls  with that of the remaining 659 unselected controls  from the pool of all patients undergoing surgery for this indication. There was no difference in gender distribution between the selected and unselected controls (P = 0.61).The prevalence of cardiovascular risk factors is summarized in Table P = 0.02). There was no difference in prevalence of former or never smokers between the groups. A trend toward a lower prevalence of diabetes mellitus was seen in cases as compared with controls .The prevalences of hypertension, hyperlipidemia, and family history of aortic aneurysms were similar in cases and controls Table . The preP = 0.56), hyperlipidemia , or family history of aortic aneurysms . The prevalence of current smokers continued to be higher even after adjustment for gender . There was a trend toward a lower prevalence of diabetes mellitus in patients with idiopathic aortitis .To evaluate whether gender differences between cases and controls were masking the differences in cardiovascular risk factors, we performed gender-adjusted analyses Table . There wP = 0.075). The median level of CRP was higher in cases among patients in whom the test was performed  versus controls . A significantly higher proportion of cases had an elevated CRP as compared with controls .The pre-operative ESR and CRP measurements in the cases and controls are presented in Table To our knowledge, this is the first study to identify risk factors for development of idiopathic aortitis. Factors independently associated with an increased risk for idiopathic aortitis discovered at the time of surgical thoracic aneurysm repair in this study were female gender and active smoking. We did not find any difference in the prevalence of hypertension, hyperlipidemia, or family history of any aortic aneurysm. We found a trend, though not statistically significant, toward a lower prevalence of diabetes mellitus in cases versus controls.The mechanism by which female gender predisposes to inflammation in the thoracic aorta is not known. The development of disease in older post-menopausal females suggests a role of sex hormones. Human aortic matrix is composed of collagen, which plays a role in load bearing, and elastin, which conveys elasticity to the aorta. Sex hormones play an important role in aortic wall compliance by regulating the elastin/collagen activity -14. 17-βWe also found active smoking to be associated with an increased risk of idiopathic aortitis. The lack of any association with former or never smoking status indicates an acute but not cumulative effect. Smoking is an important risk factor for many rheumatic diseases like lupus and increases the risk of seropositivity in rheumatoid arthritis patients, especially those who are positive for shared epitope ,18. SmokSmoking may also interact in complex mechanisms with immune response genes like human leukocyte antigen (HLA) to mediate vascular inflammation in predisposed individuals. In patients with inflammatory abdominal aortic aneurysms, active smoking and female gender were associated with high-grade tissue inflammation . HLA-DR Though tested in only a subset of patients in our study, pre-operative ESR and CRP were elevated in a higher proportion of cases than controls. However, these elevations were much lower when compared with temporal arteritis patients with aortitis whose ESR levels range from 82 to 101 mm/hour -6. It isThe strength of our study is rigorous case definition with availability of histopathology in cases and controls. The case control design allowed us to study multiple risk factors for this very rare condition. The identification of risk factors is important for therapeutic intervention. Smoking cessation may slow down the inflammatory process and consequent growth of aneurysm . AdvanceThe potential weaknesses of the study include the inclusion of surgical cases only. There may be a spectrum of disease, with mild disease not coming to medical attention. Inclusion of only cases with surgical specimens as the standard for evaluating inflammation was chosen to increase the internal validity of the study. Cases were selected from a large tertiary care referral center, perhaps introducing a potential bias for more severe disease. We have not analyzed the risk factors according to the histopathologic subsets of giant cell or lymphoplasmacytic inflammation; however, data from our retrospective cohort did not find any meaningful correlation between clinical features and histopathology due to small numbers (manuscript in preparation).Female gender and active smoking are risk factors for development of idiopathic aortitis. Future studies are needed to evaluate the utility of smoking cessation, the role of measurement of inflammatory markers, and medical treatment strategies on disease progress and outcome.CI: confidence interval; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; GCA: giant cell arteritis; HLA: human leukocyte antigen; MMP: matrix metalloproteinase; OR: odds ratio; PMR: polymyalgia rheumatica.The authors declare that they have no competing interests.VRC conceived of the study and participated in study design, data acquisition, data interpretation, and manuscript preparation. CSC carried out statistical analysis. KPL, CJM, KJW, and ELM participated in study design and data interpretation. DVM carried out the pathologic review of aortic specimens. All authors read and approved the final manuscript.


The tumour supressor gene TP53 is thought to be involved in neural apoptosis. The polymorphism at codon 72 in TP53 and the long form variants of the upstream variable number of tandem repeats (uVNTR) polymorphism in the dopamine D4 receptor (DRD4) gene are reported to confer susceptibility to schizophrenia.We recruited 934 patients with schizophrenia and 433 healthy individuals, and genotyped the locus of the TP53 codon 72 and DRD4 uVNTR polymorphisms by combining the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) with direct sequencing.No significant differences were found in the frequency of the genotype of the TP53 codon72 polymorphism between patients with schizophrenia and their controls. However, the long form alleles (≥ 5 repeats) of the DRD4 uVNTR polymorphism were more frequent in patients with schizophrenia than in controls (p = 0.001). Hence, this class of alleles might be a risk factor for enhanced vulnerability to schizophrenia . In the logistic regression analysis, the long form variants of the DRD4 polymorphism did predict schizophrenia after the contributions of the age and gender of the subjects were included , but the CC and GG genotypes of the codon 72 polymorphism of TP53 did not.The long form variants of the uVNTR polymorphism in DRD4 were associated with schizophrenia, in a manner that was independent of the TP53 codon 72 polymorphism. In addition, given that the genetic effect of the TP53 codon 72 polymorphism on the risk of developing schizophrenia was very small, this polymorphism is unlikely to be associated with schizophrenia. The roles that other single nucleotide polymorphisms (SNPs) in the TP53 gene or in other apoptosis-related genes play in the synaptic dysfunction involved in the pathogenesis of schizophrenia should be investigated. The results of neuropsychological and neuroimaging studies suggest that abnormal connections between various cortical and subcortical regions of the brain play an important role in the pathogenesis of schizophrenia ,2. DurinAmong the proteins that are related to apoptosis, the level of Bcl-2 is reduced by 25% in the middle temporal gyrus in patients with schizophrenia as compared with controls. A high Bax/Bcl-2 ratio is also detected in the neurons and glia of the temporal cortex of patients with schizophrenia , which sThe D4 dopamine receptor (DRD4) gene contains an upstream variable number of tandem repeats (uVNTR) polymorphism that comprises between two and 10 repeats. The polymorphism is located in exon 3 of the gene, which encodes the cytoplasmic portion of the receptor and is critical for its biological function ,23. AccoFor this study, 934 patients with schizophrenia  = 12.20) were recruited in Southern Taiwan from outpatient services, the intensive care unit, the community psychiatric clinic, psychiatry referrals, and the acute wards of a teaching hospital. All patients were diagnosed by senior psychiatrists in a teaching hospital according to the criteria of the Diagnostic Statistical Manual, Fourth Edition , and werDNA was extracted from the blood samples by using a DNeasy Blood and Tissue Kit  according to the instructions of the manufacturer. The extracted DNA was diluted to a final concentration of 100 ng/μl and stored at -80°C until further use.2. The primers for PCR amplification were: forward 5'-CAA CGT TCT GGT AAG GAC AA-3'; reverse 5'-GCC TAA GGG TGA AGA GGA A-3'. The following reaction conditions were used: 35 cycles of 30 s at 94°C (denaturation), 30 s at 55°C , and 30 s at 72°C (extension). The PCR products were digested with BstUI at 60°C for 2 h, and were then visualised on a 2% agarose gel that contained 0.5 μg/ml ethidium bromide. The original undigested PCR product was 488 bp in length. PCR products that corresponded to the Arg72 (CGC) variant were digested into fragments of 222 and 266 bp by BstUI, whereas the Pro72 (CCC)-specific product remained undigested. The genotypes were recorded by at least two well-trained assessors who were blinded to the clinical characteristics of the study sample.The Pro72Arg polymorphism of TP53 was analysed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR was performed using the hot-start technique in a final reaction volume of 25 μl with 1.5 mM MgClThe results of the PCR-RFLP analysis were confirmed by direct sequencing. Briefly, the PCR product was purified using a QIAquick purification column  and then used as the template for cycle sequencing with the BigDye Terminator v3.1 Cycle Sequencing Kit , according to the instructions of the manufacturer. The extended products were separated on an ABI PRISM™ 3130 Genetic Analyzer  after alcohol precipitation.t test was used to compare continuous variables between groups, and Pearson's chi-square (χ2) test was applied to the categorical variables. χ2 tests were used to test whether the genotype frequencies at each locus conformed to Hardy-Weinberg equilibrium. In addition, a χ2 test was used to determine whether intergroup differences in the genotype distributions and allele frequencies of the TP53 codon 72 and DRD4 uVNTR polymorphisms were significant between cases and controls. Moreover, a hierarchical logistic regression analysis with dummy variables was used to evaluate whether the TP53 and DRD4 polymorphisms were independent factors associated with vulnerability to schizophrenia. Possible interaction between the polymorphisms was also analysed in the logistic regression. A total of three dummy variables were created: p53CC, p53GG, and DRD4c. For the variable p53CC, a score of 1 referred to the CC genotype of the TP53 codon 72 polymorphism, whereas 0 referred to the other possible genotypes. For p53GG, a score of 1 referred to the GG genotype of the TP53 codon72 polymorphism and 0 referred to the other possible genotypes. For DRD4c, 1 referred to the long form allelic variants of the DRD4 uVNTR polymorphism (≥ 5 repeats) and 0 referred to the short form allelic variants (≤ 4 repeats). The interrelationships among age and gender of the subjects, genotype CC of the TP53 codon 72 polymorphism, long form variants (≥ 5 repeats) of the DRD4 uVNTR polymorphism, and schizophrenia were analysed by structure equation modelling with AMOS version 7.0 (SPSS). The structural equation modelling techniques used all the information that was provided by the regression techniques and path analysis. If the p value was greater than 0.05 and the adjusted goodness-of-fit index was greater than 0.9, the null model corresponded to the real structure.The variables were analysed using SPSS for Windows version 15.0 . Continuous variables were shown as means ± SD and categorical variables were presented as proportions. An unpaired In this study, the subjects comprised 934 patients with schizophrenia  and 433 controls from the community . The distributions of genotypes and alleles for the TP53 codon 72 and DRD4 uVNTR polymorphisms are shown in Table Schizophrenia is a complex trait with a strong genetic background ,30 whoseWe found that the frequency of the Pro72 variant  of the TP53 polymorphism was higher in patients with schizophrenia than in controls from the community (45.75% versus 44.03%), which was consistent with the results of a study by Chiu et al. (46.8% vs. 40.5%) . HoweverSchizophrenia is a multi-factorial genetic disease ,30 that Our results should be viewed in the light of certain limitations. Firstly, with respect to the SNPs in TP53, we only explored the association between the codon 72 polymorphism of TP53 and schizophrenia, as well as its possible interaction with long form variants of the uVNTR polymorphism of DRD4. We did not analyse other SNPs in TP53 that have been reported elsewhere to be associated with schizophrenia ,34. A reThe VNTR polymorphism in DRD4 has been reported previously to be involved in the pathogenesis of schizophrenia ,22. The The authors declare that they have no competing interests.All authors contributed to the design of the study. FW conceived the study, performed the statistical analysis, and helped to draft the manuscript. BC helped to draft the manuscript. WT carried out the molecular genetic studies and drafted the manuscript. CN, YC, and DS collected the data, coordinated the study, and helped to draft the manuscript. All authors have revised the manuscript and have approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/10/147/prepub