We sought to validate these observations in persons scheduled for coronary angiography.Heme oxygenase-1 is an inducible cytoprotective enzyme which handles oxidative stress by generating anti-oxidant bilirubin and vasodilating carbon monoxide. A (GT)We included 3219 subjects in the current analysis, 2526 with CAD including a subgroup of CAD and MI (n = 1339) and 693 controls. Coronary status was determined by coronary angiography. Risk factors and biochemical parameters  were determined by standard procedures. The dinucleotide repeat was analysed by PCR and subsequent sizing by capillary electrophoresis, the -413A>T polymorphism by PCR and RFLP.In the LURIC study the allele frequency for the -413A>T polymorphism is A = 0,589 and T = 0,411. The (GT)n repeats spread between 14 and 39 repeats with 22 (19.9%) and 29 (47.1%) as the two most common alleles. We found neither an association of the genotypes or allelic frequencies with any of the biochemical parameters nor with CAD or previous MI.n repeat or the -413A>T SNP in the HMOX1 promoter in CAD or MI.Although an association of these polymorphisms with the appearance of CAD and MI have been published before, our results strongly argue against a relevant role of the (GT) Low-grade inflammation and oxidation are important mechanisms involved in the complex pathological process of atherogenesis . SeveralIn humans two isoforms of heme oxygenase, HO-1 and HO-2, have been described. HO-1 is the inducible isoform, which is up-regulated by various exogenous stimuli, such as oxidative stress, hypoxia, or its own substrate heme ,7. The eHMOX1 promoter, a (GT) dinucleotide repeat and the -413A>T (rs2071746) single nucleotide polymorphism (SNP) are thought to be involved in the pathogenesis of cardiovascular disease by modulating the expression of HO-1 [Two different polymorphisms in the  of HO-1 -24.We investigated whether these variants are associated with angiographic coronary artery disease (CAD), myocardial infarction (MI), or mortality in a large cohort of individuals who had previously undergone coronary angiography.Ludwigshafen Risk and Cardiovascular Health (LURIC) study includes consecutive white patients hospitalized for coronary angiography between July 1997 and January 2000. The study was approved by the ethics review committee at the "Landesärztekammer Rheinland-Pfalz" . Written informed consent was obtained from each of the participants.The A detailed description of LURIC has been published . DiabeteWe included 3219 patients in this study; all of them underwent coronary angiography. To rule out racial differences as a confounding factor the LURIC study was designed to recruit probands with German ancestry only. We did not include family members of the patients for this study.Information on vital status was obtained from local person registries. No patients were lost to follow-up. Amongst the subjects included in this examination, 752 deaths (23.4%) had occurred during a median observation time of 7.97 years. Cardiovascular death (n = 473) included the following categories: sudden death, fatal myocardial infarction, death due to congestive heart failure, death immediately following intervention to treat CAD, fatal stroke and other causes of death due to CAD. All clinical assessments were made blinded to the knowledge of HMOX1 genotype.Fasting blood samples were obtained by venipuncture in the early morning. Levels of cholesterol, triglycerides, bilirubin, iron, transferrin, and ferritin were determined with common laboratory procedures . LDL-C aHindIII in the amplicon . In case of the A genotype a fragment of 163 bp and in case of the T genotype two fragments of 140 bp + 23 bp were obtained. To validate the method three samples of each genotype were sequenced; as internal control 184 DNA samples, blinded distributed within the samples, were genotyped twice. To confirm genotype assignment, the restriction fragments were analyzed independently by two scientists on two separate occasions. The results were scored blinded as to case-control status.Genomic DNA was prepared from EDTA-anticoagulated peripheral blood by using a standard salting-out procedure. Genotypes of the -413A>T polymorphism were determined using polymerase chain reaction with a mismatched 5' primer to obtain an allele specific restriction site for n repeats were each calculated using Genetic Profiler software (Amersham).The microsatellite polymorphism was amplified according to the protocol described by Kaneda et al.  and analHMOX1 alleles and genotypes in CAD+ and CAD- as well as in MI+ and MI- subjects were obtained by direct count and the deviation from Hardy-Weinberg equilibrium was examined by the chi-square test. In models assuming a co-dominant (additive) effect of the A-allele of the HMOX1 gene, the genotypes AA, AT and TT were coded as 0, 1, and 2, respectively. Genotypes were either treated as interval-scaled or categorical variables, the AA genotype being considered as the reference category in the latter case. When assuming a dominant effect, genotype TT was coded as 0, and AT and AA combined as 1, whereas assuming a recessive effect, genotypes TT and AT were coded as 0, and AA as 1. In a similar way the repeat classes  and both haplotypes  were analyzed. To examine the relationship of the genotypes with total mortality and mortality from cardiovascular causes, we calculated hazard ratios and 95 percent confidence intervals (95% CI) using the Cox proportional hazards model. The criterion for statistical significance was p < 0.05. The SPSS statistical package  was used for all statistical analyses.Clinical and anthropometric characteristics were compared between CAD patients and controls by analysis of variance (ANOVA) or logistic regression. Covariable adjustments were carried out as indicated. Associations between categorical variables were examined by chi-square testing or logistic regression analysis. The frequencies of the We included 3219 subjects in the current analysis. 693 were control subjects without CAD, 2526 were diagnosed CAD-positive by angiography and from these 1339 had suffered and survived a MI in the past. Compared to the control group, patients with CAD and with a former MI were significantly older, current or past smoker and type 2 diabetes were more prevalent  and higher triglycerides p < 0.012). No significant difference was found for the two last mentioned parameters for the CAD plus MI group According to the classification of Funk et al. , we chosChen et al.  describe2 = 0,63). Analysis of the two most common haplotypes, 22-T (19.9%) and 29-A (47.1%), did not generate new information with regard to cardiovascular disease; there was still no association with CAD or MI (data not shown).The small repeats, e.g. 22 – the most frequent ones in the class S alleles – are associated with the T allele, while the large repeats, e.g. 29, with the exception of very large repeats (34–38), are associated with the A allele .HMOX1 with mortality from all causes nor with cardiovascular mortality  had occurred during a median observation time of 7.97 years, from which 473 (14.7%) died from cardiovascular causes. Regardless whether or not we adjusted for age and gender, there was neither an association of the genetic markers of % had occHMOX1 genotypes and serum levels. However, we were not able to find any significant association between HMOX1 genotypes and serum levels of bilirubin (e.g. -413A>T and bilirubin (μmol/L): AA 10.78 ± 6.50, AT 11.12 ± 6.67, and TT 10.95 ± 5.99, means ± standard deviations) or iron, even after adjustment for age, gender, or other cardiovascular risk factors.Products of the enzymatic action of HO-1 are iron and biliverdin, which in turn is converted to bilirubin. If promoter variants influence the systemic expression of HO-1 it might be possible to detect an association between n repeat has been published [Previously a correlation of lipid parameters and the (GT)ublished . To avoin dinucleotide repeat nor the -413A>T polymorphism in the HMOX1 promoter are associated with angiographic CAD, MI or survival rate in Caucasians undergoing coronary angiography.The presented data show in a prospective case-control study of more than 3000 participants that neither the (GT)HMOX1 promoter is still under debate. Yamada et al. described a higher response of reporter gene constructs with short (GT) repeats to H202 exposure in A549 and Hep3B cell [in vitro experiments with reporter gene constructs that the length of the (GT)n repeat did not influence the activity of the HMOX1 promoter in bovine aortic endothelial cells. They found that – instead of the (GT)n repeat – the responsible element was an A>T exchange at position -413, giving rise to a 4-fold increase of activity. Therefore further experiments are required to clear the functionality of these variants.The functional relevance of both variants in the p3B cell . Similarp3B cell  or by anp3B cell . In all p3B cell ,32 showen repeat in the HMOX1 promoter in cardiovascular disease. Asian patients with a lower number of repeats were less susceptible to pulmonary emphysema and coronary artery disease [n microsatellite in the promoter of the HMOX1 gene on the risk of CAD, a history of MI, and total or cardiovascular mortality.Irrespective of the unclear functional role there are several reports suggesting a role of the (GT) disease ,18,30. F disease  suggesti disease ,22. Howe disease , who fol disease . Like thn repeat.It is not unusual, that association studies of genetic polymorphisms produce divergent results, especially if small numbers of cases and controls are examined. In many instances positive associations seen in small studies have been disproven in subsequent studies of larger cohorts. Both, our CAD and control group were substantially larger than the groups investigated in the other reports. The only report with a comparable number of participants is the study of Wijpkema et al. and their results are in line with ours . In addiConcerning the -413A>T SNP only one study has been published before  showing HMOX1 associated with a higher expression would increase bilirubin serum levels we did not see any association between HMOX1 genotypes and bilirubin concentration. We can not exclude that other genes involved in bilirubin metabolism superpose the effect of different HMOX1 genotypes. One candidate gene is UGT1A1, coding for bilirubin uridine diphosphate-glucuronosyltransferase. This enzyme is responsible for the glucuronidation of bilirubin and thus enhances its elimination. Recently, a promoter variation (UGT1A1*28) has been associated with higher bilirubin serum levels and lower risk for coronary heart disease in the Framingham Heart Study [HMOX1 genotypes and serum iron levels.On product of the enzymatic action of HO-1 is biliverdin, which in turn is converted to bilirubin. Biliverdin and bilirubin are both antioxidants, and higher serum levels of bilirubin are inversely related to the risk of CAD . Actuallrt Study . The metA methodological aspect of the present study that deserves discussion, like in many case-control association studies, is the appropriateness of the control group. Because angiography is relatively insensitive for early lesions that do not impact on the vascular lumen, early lesions undetectable by angiography could not be ruled out in controls, similar to coronary microvascular dysfunction. Nonetheless, coronary angiography remains a gold standard for the diagnosis of CAD, and for the definition of the coronary artery status, this may however be regarded as a strength of the study because of "controls" were also studied by angiography. This is crucial because the prevalence of clinically asymptomatic coronary atherosclerosis has been reported to be high at or above the age of 50 years . Hence, HMOX1 gene may be determined, at least in part, by genetics, and reduced ability to induce HO-1 expression may be involved in the mechanism of coronary atherosclerosis. Nevertheless, neither the (GT)n dinucleotide repeat nor the -413A>T polymorphism of HMOX1 gene are reliable genetic markers for cardiovascular disease in Caucasians.In conclusion, the regulation of the The authors declare that they have no competing interests.NL carried out the microsatellite analysis and contributed to the analysis and interpretation of the data as well as to the writing of the manuscript. KW participated in the design of the study and helped to perform the statistical analysis and to draft the manuscript. US and BW organised the follow-up within LURIC and contributed the survival data. BRW, BOB, and WM conceived of the LURIC study and participated in its design and coordination. MMH participated in the design of the study, carried out the SNP analysis, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


FLCN) mutations are the genetic cause of Birt-Hogg-Dubé syndrome, a monogenic disorder characterized by spontaneous pneumothorax, fibrofolliculomas, and kidney tumors. Loss-of-function folliculin mutations have also been described in pedigrees with familial spontaneous pneumothorax. Because the majority of patients with folliculin mutations have radiographic evidence of pulmonary cysts, folliculin has been hypothesized to contribute to the development of emphysema.Rare loss-of-function folliculin  and 420 male smokers with normal lung function from the Normative Aging Study (controls).None of the seven previously reported Birt-Hogg-Dubé or familial spontaneous pneumothorax mutations were observed in the 152 severe, early-onset COPD probands. Exon resequencing identified 31 variants, including two non-synonymous polymorphisms and two common non-coding polymorphisms. No significant association was observed for any of these four variants with presence of COPD or emphysema-related phenotypes.Genetic variation in folliculin does not appear to be a major risk factor for severe COPD. These data suggest that familial spontaneous pneumothorax and COPD have distinct genetic causes, despite some overlap in radiographic characteristics. FLCN) have been implicated in two genetic syndromes with shared pulmonary manifestations of spontaneous pneumothorax and lung cyst formation: Birt-Hogg-Dubé syndrome  and familial spontaneous pneumothorax [Rare mutations in the folliculin gene . FSP – dM 173600). Rare, lM 173600).BHD- and FSP-associated folliculin mutations confer distinct clinical and histopathologic pulmonary manifestations, including recurrent, idiopathic pneumothoraces ,2,7,19 aWhile these cystic changes are radiographically distinct from common forms of emphysema, increasing severity of folliculin-associated cystic changes are correlated with cigarette smoking. In addiBaseline characteristics for the cohorts are presented in Table We first assessed whether folliculin mutations known to cause either BHD or FSP were observed among 152 EOCOPD probands. We genotyped 7 previously reported folliculin mutations, of which 2 have been observed only in FSP patients, 4 have been observed only in patients with BHD-associated pneumothorax or BHD-associated lung cysts, and 1 has been reported in both conditions ; and another C to T transition at c.871+36 (rs3744124), resulting in a substitution of glycine to arginine at codon 303 in folliculin isoform 2 . Neither of these variants were predicted to have significant functional impact based on PolyPhen analysis[Although these highly penetrant mutations appear to confer distinct phenotypes of BHD and FSP but not severe COPD, it is possible that other folliculin variants could contribute to the development of EOCOPD through more subtle impact on folliculin function. To test this hypothesis, we resequenced the coding and surrounding genomic regions  of the folliculin locus in 41 EOCOPD probands (82 chromosomes) to identify potential COPD-related variation. 31 folliculin variants were identified  and two common 5' UTR variants (rs1708629 and rs1736209) – in 345 NETT subjects and 420 NAS controls and tested for evidence of genetic association with the presence or absence of COPD. We also performed population-based association analysis of quantitative emphysema phenotypes within NETT COPD cases. All variants were in Hardy-Weinberg equilibrium in the NAS controls. In keeping with the sequence analysis above, none of these four variants was associated with COPD susceptibility or emphysema phenotypes, as evidenced by nearly identical genotype distributions for all four variants between emphysema cases and healthy smoking controls , the National Emphysema Treatment Trial (NETT), and the Normative Aging Study (NAS) have been reported previously -41. Probpredicted, age < 5predicted, evidencpredicted and FEV1predicted). The NEParticipants in the Boston Early-Onset COPD Study and the NETT Genetics Ancillary study gave written informed consent. Anonymized data were used for the NAS participants, as approved by the Partners Healthcare Human Research Committee and the IRB of the VA Hospitals. The appropriate institutional review boards approved all studies.; March 2006 assembly).Seven BHD- or FSP-related folliculin mutations – including three reported in FSP, and the two most common mutations in BHD – were genotyped in 152 Boston Early-Onset COPD Study probands , for 82 independent chromosomes. Power calculations for genotyping in NETT and NAS were performed using Quanto[Statistical analysis was performed in SAS 9.1 . Baseline characteristics in NETT versus NAS and NETT versus EOCOPD subjects were compared using Fisher's exact or Wilcoxon test, as appropriate. Hardy-Weinberg equilibrium was assessed in control subjects using an exact test. Genotype frequencies were compared using Fisher's exact test. In the NETT cohort, four CT emphysema phenotypes were analyzed. Two were based on radiologist assessments: 1) upper lobe emphysema predominance and 2) overall emphysema severity on a scale of 0 to 24. Two were quantitative scores using a cutoff of -950 Hounsfield Units: 1) the difference between apical and basilar emphysema and 2) the total percentage of affected lung. The upper lobe and apical/basilar phenotypes were chosen based on the extra-apical predominance of folliculin associated lung cysts. The quantitative densitometric phenotypes were added due to reported interobserver variability in CT phenotypes. All foung Quanto, under aBHD: Birt-Hogg-Dubé Syndrome; EOCOPD: Boston Early-Onset COPD Study; FLCN: Folliculin; FSP: Familial Spontaneous Pneumothorax; MAF: Minor allele frequency; MIM: Online Mendelian Inheritance in Man; NAS: Normative Aging Study; NETT: National Emphysema Treatment Trial; UTR: Untranslated region.MHC performed the analysis and drafted the manuscript. BJK supervised the genotyping and sequencing. AAL and DS participated in study design and coordination and assisted in analysis. BAR and EKS conceived of the study and participated in its design and coordination. BAR assisted in analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Supplemental methodsClick here for file


CFH), the hypothetical LOC387715, and the high-temperature requirement A-1 (HTRA1) genes have been reported to be associated with age-related macular degeneration (AMD). The purpose of this study was to investigate the association of reported common single-nucleotide polymorphisms (SNPs) in CFH, LOC387715, and HTRA1 with exudative AMD in a northern Chinese population.Variants in complement factor H (rs1061170:T>C in CFH (Y402H), rs10490924:G>T in LOC387715 (A69S), and rs11200638:G>A in the promoter of HTRA1 was performed using a polymerase chain reaction (PCR) method followed by allele-specific restriction enzyme digestion and direct sequencing.A cohort of 121 unrelated patients with exudative AMD and 132 control subjects were enrolled in this study. Genomic DNA was extracted from blood leukocytes. Genotyping for SNPs CFH was not associated with exudative AMD in our study population. Frequencies of Y402H was 10.3% in AMD cases and 8.0% in controls (p=0.353). Significant associations were detected for exudative AMD with SNPs rs10490924:G>T in LOC387715 (A69S), and rs11200638:G>A in the promoter of HTRA1. The risk T-allele frequency of rs10490924 in LOC387715 was 64.9% in cases versus 43.2% in controls (p<0.001). The odds ratio for risk of AMD was 1.56  for the GT genotype and 5.45  for the TT genotype. The A allele frequency of rs11200638 in the HTRA1 promoter was 67.8% in cases versus 42.4% in controls (p<0.001). The odds ratio was 2.75  for the GA genotype and 7.90  for the AA genotype. An odds ratio of 7.94  was obtained for carriers with both TT genotype in LOC387715 and AA genotype in the HTRA1 promoter.The Y402H variant in LOC387715 and HTRA1 polymorphisms are associated with a higher risk of exudative AMD in northern Chinese. We found no association of CFH Y402H with exudative AMD. The low frequency of CFH Y402H variant was further confirmed in this study population.Our data suggest that the  Age-related macular degeneration (AMD) is a clinically heterogeneous disease and the leading cause of irreversible visual impairment in the elderly population worldwide ,2. The eABCA4), apolipoprotein E (APOE), and fibulin-5 (FBLN5) [AMD susceptibility is linked to both genetic and environmental factors, although its precise etiology remains elusive. Reported risk factors include ocular pigmentation, dietary factors, positive family history for AMD, smoking, and several gene mutations such as ATP-binding cassette transporter protein 4 ( (FBLN5) . Moreove (FBLN5) -14.CFH) gene on chromosome 1q31 has been demonstrated as the first major AMD susceptibility gene, and may associate with 30%–50% of AMD cases [rs1061170:T>C) and other intron variants in CFH have been reported to be associated with an increased risk of AMD in more than ten different populations of European descent [LOC387715 on chromosome 10q26 was further confirmed in this group [rs10490924:G>T in exon 1 of LOC387715 (A69S), strongly implicating this gene in the pathogenesis of AMD. Furthermore, studies of Hong Kong Chinese and Caucasian population have identified SNP rs11200638:G>A in the promoter of high-temperature requirement A-1 (HTRA1) gene, approximately 6.1 kb downstream of rs10490924 in LOC387715 on chromosome 10q26, to be associated with increased risk of AMD [Recently, the complement factor H  and 132 control subjects  participated in this study. The age range is 50 to 90 years old with mean 66.0 and standard deviation (SD) 8.4 in cases, and 50 to 84 years old with mean 66.1 and SD 6.6 in controls.All participants received a standard ophthalmic examination, including visual acuity measurement, slit-lamp biomicroscopy, and dilated fundus examination that was performed by a retinal specialist. All AMD patients had fluorescein as well as indocyanine green fundus angiography. The diagnosis of exudative AMD was based on ophthalmoscopic and fluorescein angiographic findings. Inclusion criteria for patients were as follows: 1) age of 50 years or older, 2) presence of macular lesions  in one or both eyes; and 3) macular lesions not associated with other eye diseases, such as degenerative myopia, angioid streaks, or any other retinal/choroidal diseases. Controls were confirmed not to have clinical evidence of early or late AMD in both eyes and not to have any other eye diseases aside from mild age-related cataracts. Subjects with severe cataracts were excluded from the study.2 ), 25 µM each of dNTP, 1 pmol of each primer, 0.5 units Taq DNA polymerase, and 50 ng genomic DNA. Amplification of HTRA1 rs11200638 was performed by adding 5% dimethyl sulfoxide (DMSO) to the PCR reaction mixture. Samples were denaturing at 94 °C for 5 min followed by 35 cycles under the following conditions: denature at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 45 s. The final extension step was lengthened to 5 min. Aliquots of amplified uncut products were resolved by electrophoresis in 2% (w/v) agarose gels with 0.5 µg/ml ethidium bromide and visualized under ultraviolet light. Samples were then used for allele-specific restriction enzyme digestion. Some products were also used for direct sequencing.Blood samples were collected from all participants and stored at -80 °C before DNA extraction. Genomic DNA was extracted from the peripheral venous blood for each participant using a commercially available genomic DNA extraction and purification kit  according to the manufacturer’s protocol. Genotyping was performed using a method of polymerase chain reaction (PCR) followed by allele-specific restriction enzyme digestion and direct sequencing. The primer sequences used in this study are given in CFH Y402H) or 37 °C for 3 h (PvuII for LOC387715 and EagI for HTRA1). Samples were electrophoresed on a 2% (w/v) agarose gel with 0.5 µg/ml ethidium bromide. Images of the gel were taken with a Molecular Imager Gel Doc XR System . Genotypes were determined based on the restriction patterns and were further confirmed by direct sequencing of the PCR products using an automatic ABI 3730XL DNA Analyzer  in a selected subset of subjects . Enzymes used in this study and the lengths of various restriction fragments are given in subjects .2 values in cases and controls were calculated to identify possible genotyping errors. Linkage disequilibrium (LD) was measured by the D' value using statistical software Haploview version 4.0 [t test. Genotypes and allele frequencies between cases and controls were compared using the χ2 test. Odd ratios (OR) and 95% confidence intervals (CI) were calculated according to the Woolf equation [Hardy Weinberg equilibrium (HWE) χsion 4.0 . Numericequation . A p-valt test). Percentage of males was 58.7% in the AMD group and 51.5% in the control group .Case–control samples were composed of 121 unrelated patients with exudative AMD and 132 healthy control individuals . The mears1061170:T>C in CFH (Y402H), rs10490924:G>T in LOC387715 (A69S), and rs11200638:G>A in the promoter region of HTRA1 .Genotypes were determined successfully by restriction enzyme digestion in all participants for SNPs of HTRA1 . GenotypCFH variant Y402H was not associated with exudative AMD (2 test) in the study population. No significant difference between the CFH genotypes in AMD group and control group was observed . Compared to the wild-type homozygous TT genotype, the OR for heterozygous TC genotype was 1.25 . The OR for homozygous CC genotype was not calculated since the CC homozygote was identified only in one case in this Chinese cohort.Between case participants and controls, the tive AMD . The frers10490924:G>T in LOC387715 (2 test). Genotype distributions between AMD cases and controls were statistically significantly different . Compared to wild-type GG genotype, the OR for the risk of AMD was 1.56  for the heterozygous GT genotype and 5.45  for the homozygous TT genotype.A strong association with exudative AMD was detected for SNP OC387715 . The risrs11200638:G>A in the promoter of HTRA1 was significantly associated with exudative AMD (2 test). Genotypes of this HTRA1 promoter polymorphism between AMD cases and controls were statistically significantly different . The OR was 2.75  for the heterozygous GA genotype and 7.90  for the homozygous AA genotype, compared to the wild-type GG genotype.Similarly, the SNP tive AMD . FrequenLOC387715 rs10490924 and HTRA1 rs11200638 genotype combination frequencies in patients with exudative AMD and control subjects are shown in LOC387715 and AA genotype of HTRA1  and in controls .The of HTRA1 . LOC3877CFH (rs1061170) has been identified as a common nonsynonymous variant and a major genetic risk factor for AMD development in Caucasian populations. Frequencies of the risk C allele in Caucasians were between 61%–94% in AMD and 34%–46% in controls [The missense polymorphism Y402H in controls -21. In ccontrols ,31. In Hcontrols . In Taiwcontrols  found nocontrols ,31. Sevecontrols -35. Dramcontrols .CFH is involved in the regulation of the alternative complement pathway. Complement components have previously been found in drusen, an inflammatory deposit between retinal pigment epithelium and neuron-sensory retina, suggesting dysregulation of complement activation may be involved in AMD development [CFH Y402H variant to C-reactive protein was strongly reduced compared to the wild-type, indicating that the association of the CFH Y402H with AMD could be due to reduced clearance of cellular debris and increased local inflammation. Magnusson et al. [CFH Y402H variant have been reported in Japanese populations [elopment . Recentlelopment  reportedn et al.  demonstrn et al. . The lowulations -35,42,43ulations -46.CFH Y402H variant, our data demonstrate strong associations for the risk of exudative AMD with LOC387715 and HTRA1 variants. Consistent with previous published findings for the Hong Kong Chinese [LOC387715 rs10490924 or the A allele of rs11200638 in the promoter region of HTRA1. This finding is also consistent with those of Japanese studies published recently [LOC387715 and HTRA1 genes associate with susceptibility to AMD development across diverse ethnicities. In this study, LOC387715 rs10490924 and HTRA1 rs11200638 showed similar significance levels. ORs for exudative AMD were 1.56  for the heterozygous GT genotype and 5.45  for the homozygous TT genotypes of LOC387715 rs10490924, when compared to the wild GG genotype. ORs for exudative AMD with heterozygous GA genotype and homozygous AA genotype of rs11200638 in the promoter of HTRA1 were 2.75  and 7.90 , respectively, when compared to the wild-type GG genotype. Individuals with both TT genotype of LOC387715 and the AA genotype of HTRA1 associated with an OR of 7.94 , similar to the risk conferred by rs10490924 or rs11200638 alone. Based on the likelihood ratio test, no interaction or combined effect was evident between the two SNPs: rs10490924 and rs11200638.In contrast to  Chinese  and Cauc Chinese ,47-49, orecently -52. The LOC387715 and HTRA1 are both located on the chromosome 10q26 region, which is one of the major chromosomal regions identified for AMD susceptibility. LOC387715 encodes a hypothetical protein of unknown function and is highly expressed in placental tissue, but its expression in the human retina is weak [rs10490924 maps to exon 1 of the hypothetical LOC387715 gene and changes putative amino acid 69 from alanine to serine (A69S). HTRA1 encodes a heat shock serine protease and is expressed in the mouse and human retina [rs11200638 resides in the promoter of HTRA1 and is approximately 6.1 kb downstream of the LOC387715 rs10490924. Because only a single cDNA sequence of LOC387715 has been found, DeWan and co-authors [rs10490924 is a surrogate marker that is correlated, or is in LD, with the putative AMD disease-causing variant. They thus concluded that HTRA1 is a major risk factor for exudative AMD. Contrary to the reports of DeWan et al. and others [LOC387715, but not HTRA1, represents a major susceptibility variant for AMD at 10q26. In their study, Kanda and coauthors [rs11200638 has no significant impact on HTRA1 promoter activity in three different cell lines, and HTRA1 mRNA expression exhibits no significantly different change between control and AMD retinas. However, it has been demonstrated that LOC387715 mRNA is detected in the human retina and various cell lines and encodes a 12 kDa protein, which localizes to the mitochondrial outer membrane when expressed in mammalian cells [LOC387715 rs10490924 and HTRA1 rs11200638 are in high LD. Further studies are needed to clarify whether LOC387715 rs10490924 and HTRA1 rs11200638 are only in LD or are causative factors for AMD. is weak . The risd others , a studyd others  showed toauthors  showed tan cells . Kanda aan cells  thereforLOC387715 and HTRA1, but not the CFH Y402H polymorphism, conferred significantly increased risk for exudative AMD in a northern Chinese population. Replication of association studies in diverse ethnic groups worldwide may provide a better appreciation of the genetic contributions in AMD pathogenesis. Further studies would be needed to determine the identity of causal variant and to evaluate the possible mechanisms through which the variant influences the disease susceptibility.In summary, our data demonstrated that


Bladder cancer has been linked with long-term exposure to disinfection by-products (DBPs) in drinking water.S-transferase  and cytochrome P450 (CYP2E1) genes in the metabolic pathways of selected by-products on bladder cancer in a hospital-based case–control study in Spain.In this study we investigated the combined influence of DBP exposure and polymorphisms in glutathione GSTT1, GSTZ1, and CYP2E1 polymorphisms on bladder cancer using adjusted logistic regression models with and without interaction terms.Average exposures to trihalomethanes  from 15 years of age were estimated for each subject based on residential history and information on municipal water sources among 680 cases and 714 controls. We estimated effects of THMs and GSTT1 +/+ or +/− versus GSTT1 null (pinteraction = 0.021), GSTZ1 rs1046428 CT/TT versus CC (pinteraction = 0.018), or CYP2E1 rs2031920 CC versus CT/TT (pinteraction = 0.035). Among the 195 cases and 192 controls with high-risk forms of GSTT1 and GSTZ1, the ORs for quartiles 2, 3, and 4 of THMs were 1.5 (0.7–3.5), 3.4 (1.4–8.2), and 5.9 (1.8–19.0), respectively.THM exposure was positively associated with bladder cancer: adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were 1.2 (0.8–1.8), 1.8 (1.1–2.9), and 1.8 (0.9–3.5) for THM quartiles 2, 3, and 4, respectively, relative to quartile 1. Associations between THMs and bladder cancer were stronger among subjects who were Polymorphisms in key metabolizing enzymes modified DBP-associated bladder cancer risk. The consistency of these findings with experimental observations of GSTT1, GSTZ1, and CYP2E1 activity strengthens the hypothesis that DBPs cause bladder cancer and suggests possible mechanisms as well as the classes of compounds likely to be implicated. Chlorine is a cost-effective drinking water disinfectant that has been used since the early twentieth century to control a panoply of waterborne infectious diseases. By-products of the interaction of chlorine with organic precursors in water were first noted in 1974, with the discovery of trihalomethanes (THMs) in disinfected water . Since tS-transferase (GST) theta-1 (GSTT1) activates brominated THMs to mutagens in a transgenic strain of Salmonella  in GSTZ1 and CYP2E1 resulting in modified enzymatic activity , Alicante, and Tenerife] . EligiblControls were selected from patients admitted to participating hospitals with conditions thought to be unrelated to the major risk factors of bladder cancer, such as tobacco use. Diagnostic categories for controls were as follows: 37% hernias, 11% other abdominal surgery, 23% fractures, 7% other orthopedic problems, 12% hydrocoele, 4% circulatory disorders, 2% dermatological disorders, 1% ophthalmological disorders, and 3% other diseases. Controls were individually matched to cases by age at interview within 5-year strata, sex, ethnic origin, and hospital catchment area, a well-defined area corresponding to the specific health services region covered by each hospital. Written informed consent was obtained from each subject before the study. The study was approved by the review board of each participating institution and in accord with an assurance filed with and approved by the U.S. Department of Health and Human Services.n = 1,219) and 87% of controls  participated. Subjects who refused to answer the CAPI were administered a reduced interview of critical items (21% of cases and 19% of controls). Questionnaire information on water-related exposures used in this analysis included residential history from birth  and swimming pool use as an adult. These data were collected from all participants, including those who responded to the critical items questionnaire.After obtaining informed consent, trained interviewers administered a computer-assisted personal interview (CAPI) to participants during their hospital stay. Interview items included sociodemographic characteristics; smoking habits; occupational, residential, and medical histories; and familial history of cancer. We identified 1,457 eligible cases and 1,465 eligible controls. Of these, 84% of cases  measured levels of the four THMs  in 113 tap water samples from the studied geographic areas between September and December 1999.Average THM levels in recent years were extrapolated back to approximately 1920. Historical THM levels were estimated by municipality under the assumption that past THM levels were similar to current concentrations when the water source had not changed. When the water source had changed, we calculated the average THM level using the proportion of surface water during the relevant time period. We assumed that the THM level before the start of chlorination was zero. The exposure assessment was described previously , 2007.We merged individual and municipal databases with individual residential information by year and municipality, and obtained individual year-by-year average THM levels, water source, and chlorination status for each study subject. We used as an exposure metric the average THM level of the water source serving participant residences in the period between age 15 years and the interview, as described previously in the analysis of the main effects of DBP exposure . As in tGSTT1, GSTM1, and N-acetyltransferase 2 (NAT2) were conducted at the Core Genotyping Facility of the Division of Cancer Epidemiology and Genetics . The SNPs of NAT2 and assignments of rapid/intermediate/slow acetylator types, as well as the GSTM1 genotypes, have been described in detail elsewhere (GSTT1 genotypes were defined as null (−/−) if a deletion was found in both copies of the gene and present if one (+/−) or none (+/+) of the copies had a deletion. We used the TaqMan assay  for SNPs in NAT2, two of three SNPs in GSTZ1 [E32K (rs7975) and G42R (rs7972)], and deletions in GSTT1 and GSTM1. The methods for each specific assay are available from the GSTZ1 (T82M rs1046428) and three SNPs in CYP2E1  were determined in a GoldenGate assay  (n = 93 pairs) showed ≥ 99% agreement for all assays.DNA for genotype assays was extracted from leukocytes or mouthwash samples as described previously . Genotyplsewhere . GSTT1 gCA, USA) . All genp-trends were calculated using a likelihood ratio test comparing the model with and without the THM exposure variable modeled as a continuous variable with each quartile coded according to its median value.We considered results to be statistically significant if the alpha level was ≤ 0.05. Subjects were grouped by quartile of the time-weighted average THM level since age 15 years. We used unconditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs). ORs were adjusted for age (continuous), sex, smoking status (never/former/current), size of the municipality of longest residence until 18 years of age , education , geographic area , and overall quality of interview . Analyses using more detailed smoking information  gave similar results and are not reported. Subjects were defined as “former” smokers if they had quit at least 1 year before the interview. Missing data for covariates were coded to a separate category for each variable included in the models. This applied to a small proportion of respondents for each covariate. For example, < 1% of respondents were missing data on smoking status . Linear GSTZ1 and CYP2E1 were estimated using HaploStats  using the program language R (http://www.r-project.org/).Interactions between genotypes and exposure to DBPs, as estimated by average THM levels serving the household between 15 years of age and diagnosis (cases) or interview (controls), were also tested using the likelihood-ratio test to allow estimation of parameters under the assumption of genotype–DBP (THMs) independence in the source population. Tests for multiplicative interaction were used to assess whether the genotype ORs within categories of DBP exposure or, equivalently, DBP ORs within genotype categories differed significantly from each other. Haplotype frequencies for Of the 1,219 cases and 1,271 controls interviewed, 1,188 (97%) cases and 1,173 (92%) controls provided a blood or buccal cell sample for DNA extraction. After excluding cases and controls with low amounts of DNA, nonwhite individuals (to limit heterogeneity), and DNA quality control difficulties, the study population with adequate genetic material available for analysis numbered 1,150 cases and 1,149 controls . After fptrend = 0.029) (NAT2 slow acetylator compared with rapid/intermediate genotypes [OR (95% CI) of 1.4 (1.2–1.7)] and GSTM1 null (−/−) versus GSTM1 present genotypes [1.7 (1.4–2.0)]. Estimates in the present analyses were similar, with NAT2 (slow compared with rapid plus intermediate acetylator type) having an OR (95% CI) of 1.33 (1.06–1.68), and with GSTM1 null compared with the GSTM1 present genotype having an OR (95% CI) of 1.77 (1.41–2.22). We found slightly elevated, nonsignificant risks for GSTT1 presence compared with null [1.21 (0.92–1.59)]. The association for the inverse relationship (null vs. presence) was 0.81 (0.61–1.09). In the full study, compared with the homozygous presence of GSTT1, the ORs (95% CIs) were 1.2 (1.0–1.5) for heterozygotes (+/−) and 1.0 (0.8–1.3) for homozygous null (−/−) (The ORs (95% CIs) for former [2.7 (1.9–3.7)] and current [5.9 (4.2–8.5)] smokers of cigarettes in the analysis data set, relative to never-smokers, paralleled those found in the full study population . The ORs= 0.029) , similarll (−/−) .GSTZ1 and the three variants of CYP2E1 that we tested. Associations for the three SNPs in GSTZ1 and the three in CYP2E1 have not been reported previously. The GSTZ1 SNPs were in linkage disequilibrium ; however, they were not highly correlated with each other (r2 ranged from 0.02 to 0.18). Of the three CYP2E1 SNPs tested, rs8192766 was not in linkage disequilibrium with rs2070676 (D′ = 0.34). The other two pairs were in linkage disequilibrium (D′ ≥ 0.98). None of the three CYP2E1 SNPs was highly correlated with any other (r2 ranged from 0.006 to 0.34). Haplotype analysis for GSTZ1 and the three SNPs genotyped revealed no common haplotype significantly associated with risk . We made similar observations for CYP2E1 .We found nonsignificant elevations or decreases in risk for the three functional SNPs of GSTT1 (present vs. null) (pinteraction = 0.021), exon 7 +29T>C rs1046428 in GSTZ1 (pinteraction = 0.018), and −1054C>T rs2031920 in CYP2E1  or for the two other SNPs tested in CYP2E1 . We found no significant trends in associations with increasing THM quartiles among subjects with the GSTT1 null, GSTZ1 CC, or CYP2E1 CT/TT genotypes; however, we did find significant positive trends with increasing THM exposure among subjects with the GSTT1 present genotype GSTZ1 CT/TT, or CYP2E1 CC . We found no evidence of interaction between either NAT2 slow acetylator or GSTM1-null genotype and long-term average THM level .We observed significant interactions with THMs for YP2E1 CC . HaplotyCYP2E1 rs2031920 (no subjects carried the TT variant), it was not possible to analyze a three-way interaction that included these subjects. We found a significant interaction between average THM level and combined GSTT1 and GSTZ1 rs1046428 genotypes (pinteraction = 0.0052) in analyses that included subjects with both the variant and common forms of CYP2E1 rs2031920  in the highest quartile of THMs (ptrend = 0.0012). In contrast, among subjects with both GSTT1 null and GSTZ1 CC genotypes, we found no increase in relative risk with increasing THM level (pinteraction = 0.0052). These analyses excluded subjects with both low-risk variants of GSTZ1 and high-risk variants of GSTT1, or vice versa. When we restricted this analysis to subjects with the common form (CC) of CYP2E1 rs2031920, among those with both GSTT1 present and GSTZ1 CT/TT genotypes, the OR increased monotonically to 9.3 (2.5–34.0) in the highest quartile of THM exposure (> 49.0 μg/L), relative to participants with these genotypes in the lowest quartile .Because of small numbers of subjects  with the CT variant of s2031920 . Among sGSTT1 present, GSTZ1 rs1046428 CT/TT, and CYP2E1 rs2031920 CC. Among subjects with exposure to THMs in the highest quartile, the ORs were elevated and the 95% CIs excluded 1.0 for GSTT1 present (vs. GSTT1 null), GSTZ1 (CT/TT vs. CC), and CYP2E1 (CC vs. CT/TT), whereas ORs for these polymorphisms were close to 1.0 among subjects in the lowest THM exposure quartile. The associations with bladder cancer risk for NAT2 slow versus rapid/intermediate acetylator and GSTM1 null versus present within each THM stratum were variable and consistent with the overall elevated main effects for these genes.We calculated the main effects for the respective SNPs within increasing quartile strata of long-term average THMs  where weGSTT1 present (vs. GSTT1 null) and with GSTZ1 CT/TT (vs. CC) were in a direction contrary to expectation, given what we observed for interactions with THMs .In earlier analyses of these data, we found a significant association with ever swimming in pools [OR = 1.62 ] but no association with increasing hours of lifetime pool use . There wGSTT1 and GSTZ1 code for enzymes in the respective biotransformation pathways of two groups of DBPs, the brominated THMs and the α-haloacids. CYP2E1 oxidizes THMs and likely many other compounds in the DBP mixture. Overall, without considering DBP effects or interaction, we found a weak, nonsignificant overall association between polymorphisms in each of these genes and bladder cancer risk. Associations between increasing quartiles of THMs and bladder cancer were stronger among subjects with GSTT1 +/+ or +/− versus −/−, GSTZ1 rs1046428 CT or TT versus CC, and CYP2E1 rs2031920 CC versus CT (no subjects had TT), with statistically significant interactions for each of the respective gene variants. Among the 195 cases and 192 controls with GSTT1 present and GSTZ1 rs1046428 CT/TT, ORs (95% CIs) for quartiles 2, 3, and 4 of long-term average THMs were 1.5 (0.7–3.5), 3.4 (1.4–8.2), and 5.9 (1.8–19.0), respectively, relative to quartile 1. We also found that main effect estimates for bladder cancer in association with these polymorphisms varied by THM level, with significant associations for all three high-risk genotypes among subjects within the highest quartile of long-term THMs  involved. Dibromonitromethane, a DBP that has not been tested for carcinogenicity, is also activated to a mutagen by a transgenic strain of Salmonella expressing GSTT1-1  and the highest peak levels of mutagenicity in their urine because of unmetabolized urinary BDCM  or interview (controls). We included 680 cases  and 714 controls , and associations between bladder cancer and cigarette smoking, long-term exposure to THMs, and the main effects of nd GSTM1  were comBladder cancer risk has been consistently linked with long-term exposure to DBPs, a complex mixture of halogenated compounds in drinking water created when chlorine is used as a disinfectant . Experim


Genotype-phenotype association studies are typically based upon polymorphisms or haplotypes comprised of multiple polymorphisms within a single gene. It has been proposed that combinations of polymorphisms in distinct genes, which functionally impact the same phenotype, may have stronger phenotype associations than those within a single gene. We have tested this hypothesis using genes encoding components of the renin-angiotensin-aldosterone system and the high blood pressure phenotype.Our analysis is based on 1379 participants of the cross-sectional SUNSET study randomly selected from the population register of Amsterdam. Each subject was genotyped for the angiotensinogen M235T, the angiotensin-converting enzyme insertion/deletion and the angiotensin II type 1 receptor A1166C polymorphism. The phenotype high blood pressure was defined either as a categorical variable comparing hypertension versus normotension as in most previous studies or as a continuous variable using systolic, diastolic and mean blood pressure in a multiple regression analysis with gender, ethnicity, age, body-mass-index and antihypertensive medication as covariates.Genotype-phenotype relationships were explored for each polymorphism in isolation and for double and triple polymorphism combinations. At the single polymorphism level, only the A allele of the angiotensin II type 1 receptor was associated with a high blood pressure phenotype. Using combinations of polymorphisms of two or all three genes did not yield stronger/more consistent associations.We conclude that combinations of physiologically related polymorphisms of multiple genes, at least with regard to the renin-angiotensin-aldosterone system and the hypertensive phenotype, do not necessarily offer additional benefit in analyzing genotype/phenotype associations. Genotype-phenotype association studies have become important tools to explore the pathophysiology of many disease states. They are typically based on single polymorphisms in genes of interest. In some cases multiple polymorphisms within a given gene exist in a fixed combination, i.e. as haplotypes which may exhibit stronger/more consistent associations with phenotypes than single polymorphisms ,2.An expansion of this thought has been based upon studies in the renin-angiotensin-aldosterone (RAAS) system. The RAAS is an important regulator of cardiovascular function and blood pressure ,4. It coAccordingly, numerous studies have tested whether any of the above three polymorphisms is associated with the presence or severity of arterial hypertension (HT), almost all of them using the categorical variable HT rather than the underlying continuous variables of measured blood pressure. However, the available data remain equivocal as reports of associations have not been consistently confirmed, and even reports on inverse associations have been published ,11. RetrOur analysis is based on data obtained from the SUNSET study . In 1975, almost half the population of the former Dutch colony Surinam migrated to the Netherlands. Approximately 80% of these immigrants are black  or South Asian . SUNSET is based on a random sample of 2975 individuals, aged 35-60 years, drawn from the Amsterdam population register, as previously described . For furBlood pressure was measured in the morning on the right arm in a seated position with a validated, automated, oscillometric, digital device  by trained staff using appropriate cuff sizes. Two readings were taken after the subject had emptied the bladder and had been seated for at least 5 min; the mean of the two readings was used in the analyses . In lineDNA was isolated from whole blood using an Autopure LS . The AGT M235T (rs699), ACE I/D (rs4340) and AGTR1 A1166C (rs5186) polymorphisms were genotyped as previously described .Allele frequency was calculated in Haploview version 3.11 . FRP anahttp://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3/.Data on continuous variables are shown as means ± SD or as parameter estimates with 95% confidence intervals. Categorical variables such as the prevalence of alleles and FRPs as well as the prevalence of HT are reported as proportions (%). Differences in prevalence of HT between groups defined by genotype were tested using Fisher's exact test Table . Multipl2. Mean SBP, DBP and MAP were 126.3 ± 19.7 mmHg, 82.0 ± 11.6 mmHg and 96.8 ± 13.8 mmHg, respectively, with 15.6% of all subjects reporting use of antihypertensive medication. According to our categorical definition 451 subjects (32.7%) were hypertensive. The genotype prevalences are shown in Table The mean age of the study population  was 45.4 ± 6.7 years with 41.6% being male. The BMI was 27.3 ± 5.2 kg/mWhile the prevalence of HT did not differ significantly between carriers of the AGT or ACE alleles, a significantly higher HT prevalence was found in A allele carriers of the AGTR1 (33.6% vs. 27.1%; Table Linear regression models using gender, ethnicity, age, BMI and concomitant antihypertensive medication as covariates did not detect statistically significant or clinically relevant associations of AGT or ACE alleles with SBP, DBP or MAP Table . ConsequAs AGT, ACE and AGTR1 act in concert, it has been proposed that combinations of polymorphisms in the corresponding three genes may exhibit stronger/more consistent associations with phenotypes related to renal function than those in each of the three genes studied in isolation ,18. The Our analysis originates from a population-based sample rather than a case-control design. This implies that only a fraction of all participants were hypertensive and that a fraction of the HT subjects were currently receiving antihypertensive treatment. A detailed description of this population has been presented previously -23.While the PHASE program should also be suitable for FRP analysis on mathematical grounds, it should be pointed out that it was neither designed nor validated for such analysis.The detection of genotype-phenotype associations may be complicated by various factors including ethnicity but despite a large number of previous studies there is no consistent evidence for ethnicity-dependent associations of RAAS component polymorphisms with HT ,11. As sTo increase the robustness of our analysis ,30, we hIn our data set the M235T SNP in the AGT gene or the ID polymorphism in the ACE gene did not significantly associate with the blood pressure/hypertension phenotype. This is in line with the overall picture emerging from many previous studies looking at these polymorphisms -15. WhilThe main question of our analysis was whether the use of FRPs allows detection of stronger and/or more consistent detection of genotype/phenotype associations. With regard to the categorical variable HT, FRPs were only associated if they included the A allele of the AGTR1 gene. In these cases the prevalence of HT was rather similar in FRPs containing the A allele as compared to A allele carriers studied irrespective of their genotype for the two other genes. A more complex situation was found for the continuous phenotypic variables SBP, DBP and MAP. For these three variables the use of FRPs yielded statistical significance of genotype/phenotype associations in some cases where that was not found for the A allele studied in isolation. On the other hand, apparent effect sizes of the A allele were smaller when viewed in the context of FRPs than when looking at the A allele in isolation. Reaching statistical significance despite smaller group and smaller effect sizes indicates that the use of FRPs has yielded more consistent subpopulations with smaller intra-group variance.At least for the high blood pressure phenotype, our data do not support the previously proposed concept that FRPs yield stronger genotype/phenotype associations than individual polymorphisms of components of the RAAS. However, they may yield more homogenous patient groups, a possibility to be confirmed in future studies in independent populations. FRPs containing not only genes within the RAAS but also those of effectors of the RAAS, e.g. those involved in sodium and water handling, may also be worthwhile exploring. However, a more in-depth analysis of haplotypes within single genes and or the search for rare mutants with severe consequences may be a more promising approach.ACE: angiotensin-converting enzyme; AGT: angiotensinogen; AGTR1: angiotensin II type 1 receptor; DBP: diastolic blood pressure; FRP: functionally related polymorphism; HT: hypertension; I/D: insertion/deletion; MAP: mean arterial pressure; RAAS: renin-angiotensin-aldosterone system; SBP: systolic blood pressure; SNP: single nucleotide polymorphismThe authors declare that they have no competing interests.IGMvV and RPK were involved in the design of the SUNSET study. GM was involved in sample collection. INH, RPK and MCM were involved in the design of this analysis. All authors contributed to writing the manuscript, which has primarily been drafted by INH and MCM.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/10/23/prepub


Mendelian randomization refers to the random allocation of alleles at the time of gamete formation. In observational epidemiology, this refers to the use of genetic variants to estimate a causal effect between a modifiable risk factor and an outcome of interest. In this review, we recall the principles of a “Mendelian randomization” approach in observational epidemiology, which is based on the technique of instrumental variables; we provide simulations and an example based on real data to demonstrate its implications; we present the results of a systematic search on original articles having used this approach; and we discuss some limitations of this approach in view of what has been found so far. During the past decade however, the validity of observational studies has been questioned . This isetc.) are complex and are influenced by multiple risk factors that may be correlated with each other. In this context, each factor is expected to have a small influence on disease risk. Epidemiologists have the hard task to determine whether a putative risk factor is causally related to a specific disease, independently of all other risk factors. A promising approach to help epidemiologists in this task is Mendelian randomization. In this review, we first recall the principles of a “Mendelian randomization” approach in observational epidemiology (Section 2), we then provide some technical explanation of the method of instrumental variable (Section 3), followed by simulations and an example with real data (Section 4). We then present the results of a systematic search on original articles having used this approach (Section 5), discuss its limitations (Section 6) and present concluding remarks (Section 7).One cannot, for ethical and technical reasons, randomize risk factors using controlled trials in humans. The identification of risk factors therefore relies on observational studies, which are prone to spurious results due to confounding factors, reverse causation, and/or selection biases . As a co2.Mendelian randomization refers to the random allocation of alleles at the time of gamete formation. A specific genotype carried by a person therefore results from two such randomized transmissions, one from the paternally inherited allele and the other from the maternally inherited allele. A logical consequence of these randomizations is that genotypes are not expected to be associated with known (measurable or not) or unknown confounders for any outcome of interest, except those lying on the causal pathway between the genotype and the outcome. This should hence allow analyzing the genotype-risk factor association and the genotype-outcome association in an unconfounded manner. By combining appropriately the results of these two analyses, one can get an estimate of the risk factor-outcome association, which is itself not confounded. This is analogous to randomized controlled trials (of sufficient sample size), in which the random allocation of treatment (or preventive measure) is expected to lead to an even distribution of (known or unknown) confounding factors across each groups. The term “Mendelian randomization” is now frequently used in observational epidemiology to refer to the use of genetic variants to estimate a causal effect between a specific modifiable risk factor and a trait/disease of interest. The idea is to overcome some of the problems encountered in observational epidemiology, such as residual confounding and reverse causation, by taking advantage of the natural random allocation of alleles during meiosis .ALDH2) gene encodes the enzyme aldehyde dehydrogenase, which catalyzes the chemical transformation from acetaldehyde to acetic acid. Carriers of the ALDH2 *2*2 genotype have reduced alcohol consumption because of adverse reactions  due to acetaldehyde accumulation. This fact has been used to show that alcohol intake increases the risk of esophageal cancer [ALDH2 genotypes can be measured accurately, are present since birth, result from the random allocation of the paternally and maternally inherited alleles, are strongly associated with alcohol consumption, and therefore provide a unique opportunity to assess, in an unconfounded manner, the risk of disease associated with alcohol consumption. As we shall discuss in Section 6, such an approach - although appealing - also raises some methodological issues.We here provide an example to illustrate this approach. The aldehyde dehydrogenase 2 . When observing such an association in epidemiological research, however, it is often difficult to determine which of the two variables (X or Y) is the cause and which the effect, or whether a third variable  related to both variables is responsible for the observed association. Moreover, measurement error could attenuate the beta coefficient. Thus, it is not obvious how a significant non-zero  beta coefficient obtained from a classical (ordinary) least squares estimate should be interpreted. Here are five possible interpretations (among many others):X on Y.The beta coefficient is a consistent estimate of the causal effect of X on Y because of measurement error.The beta coefficient is actually underestimating the true causal effect of X on Y because of the presence of a confounder which is positively related to both X and Y.The beta coefficient is overestimating the true causal effect of X and Y: in fact there is no causal effect of X on Y.The non-zero beta coefficient is entirely due to the presence of a confounder which is related to both Y on X, not of X on Y .The beta coefficient is non-zero because of a causal effect of We consider the case where an association between a continuous (or binary) modifiable exposure X on Y”, i.e., the effect that would be observed if one could intervene and change someone’s X level by one unit, leaving other characteristics unchanged, no definitive conclusion can be drawn from such an analysis. We shall see below, illustrated in the context described by In other words, if the interest lies in assessing “the causal effect of β1 is the causal effect of X on Y and where γ1U plays the role of the error term, U being some unobserved confounder. Whenever X is correlated with the error term  Z is correlated with X, (2) Z is independent from U, and (3) Z and Y are independent given X and U. Note that the former of these conditions is verifiable from the data, whereas the latter two are largely not.The method of instrumental variables has been proposed to correct for the bias of the least squares estimate. For this, we need to have at our disposal an “instrumental variable”, or instrument γ2U plays the role of the error term in the model. Since Z is by assumption uncorrelated with this error term, the coefficients of this second model are estimated without bias by least squares. Note that the first model can be rewritten as:A second linear model  estimate of the causal effect β1 in the first model is the quotient of the two least squares estimates of slope parameters β3 and β2 in the third and second models. Since the expectation of a quotient of two estimates is asymptotically equal to the quotient of the expectations of these estimates, the IV estimates are asymptotically unbiased, but they may be biased in finite samples.At the end, the parameters of the first model can be consistently estimated using relationships β1 in the first model . The standard error of the estimates will depend, among others, on the percentage of explained variance in the second model (itself related to the percentage of explained variance in the third model). If this percentage is low, the instrument is said to be weak, the standard errors will be large and the test above will have low power. Moreover, the bias of the IV estimates is typically larger, and the asymptotic normal distribution of the IV estimates may be a poor approximation to the true distribution, when the instrument is weak, the inference being then unreliable [β2 in the second model is inferior to 10 [Asymptotically, the IV estimates are normally distributed and explicit formulae for the standard errors are available, enabling to calculate confidence intervals and to test for the nullity of the causal effect reliable . In pracor to 10 .X on Z in a first stage (this is the second model above), and regressing Y on the obtained fitted values X̂(Z) in a second stage. The method of instrumental variables can be readily extended to the case of several instrumental variables , which may be useful to improve the precision of the instrumental variable estimate. One can also adjust for additional covariates in each of the above models.Another equivalent way to calculate the IV estimates (but without their standard errors!) is to perform a “two-stage least squares”, regressing β1, one may also test for the absence of correlation between X and the error term in the first model, implying the equality of the parameters β1 and In addition to test for the nullity of the causal effect 4.Z satisfying the conditions. For simplicity, we took all intercepts in these models to be 0, all slopes to be 1, and the variables which were generated at each step were taken to be N, i.e., normally distributed with mean 0 and variance 1.To illustrate that the method of instrumental variable is effective, we simulated data from five models consistent with the five above-mentioned interpretations . In eachX on Y that we are looking for is β1 = 1 under the first three models, and is β1 = 0 under the last two models. Boxplots of the least squares (LS) estimates and of the instrumental variable (IV) estimates of parameter β1 obtained from 1,000 samples of size n = 100 under each of the five models are shown on the top panel of The causal effect of Z were set to 0.25 (instead of 1) in each model. In addition, we reduced the sample size to n = 25. Under that setting, the F-statistic in the first stage regression was smaller than 10 in more than 95% of the generated samples. Boxplots of the estimates obtained from 1,000 samples are shown on the bottom panel of To provide an idea of what may happen when using a weak instrument, we considered the same five models, but the slopes involving GGT1) gene as well as circulating GGT levels (X). CoLaus participants have been genotyped using the Affymetrix 500 K chip, alcohol consumption was assessed using a standardized questionnaire and coded in units of alcohol per week, and GGT levels were measured using standard procedures as previously described [Y to be the reported alcohol consumption and tested whether circulating GGT (X) could cause alcohol consumption  using the best GGT1 marker as our instrument (Z). Rs2017869 explained 1.12% of circulating GGT levels. The parameter β1 was estimated using least squares and the method of instrumental variables (the latter with the ivregress 2sls command implemented in Stata 10.0). The LS estimate (95%CI) was 5.53  mmol/L per risk allele. The IV estimate (95%CI) was −4.60  mmol/L per risk allele, which was significantly different from the LS estimate in a Durbin-Wu-Hausman test (P = 0.03), and not significantly different from zero. Thus, while the result provided by least squares was highly significant, the instrumental variable approach did not show any evidence for a positive causal association of GGT on alcohol consumption.We next provide an example with real data to illustrate that the method of instrumental variable is able to correct for the bias of least squares in a case of reverse causation. We used the 1,268 participants of the population-based CoLaus study , who repescribed . As we w5.We searched MEDLINE using the following «Mendelian randomization» OR “Mendelian randomisation”, which retrieved 99 citations . We acknowledge that this search strategy might not have retrieved all publications using the concept of Mendelian randomization, but it should provide a good overview of what has been published. The aim was to identify original articles reporting results from an observational study using a Mendelian randomization approach. We also searched references from review papers and original articles, as well as citations of these papers.i.e., to confirm or disprove causality). Yet, what is of interest in the Mendelian randomization approach is to estimate the causal effect of X, the modifiable factor, on Y and not simply the association between Z and Y.We identified 23 studies with a dichotomous trait as the outcome of interest  and 15 s6.et al.l these conditions for a given risk factor—outcome association. To fulfill the first condition, Z and X should be correlated . This indirectly implies that there is some level of allelic homogeneity . Note that for many exposures, no suitable genetic instrument is available. The second and third conditions are the problematic ones. They state that Z is  independent from all potential confounders U, and that Z and Y are independent conditionally on X and U [i.e., the law of independent assortment of alleles at the time of gamete formation) is not always true in that genetic variants located on the same chromosome, particularly for close loci, do not segregate independently , as detailed in Lawlor et al. [i.e., correlations between alleles at nearly loci. In genetic epidemiology, the second condition implies, among others, that there should be no confounding due to linkage disequilibrium  [Z does not necessarily need to be causally associated with X, in that another genetic variant associated to both Z and X might be the true causal variant [i.e., the existence of population subgroups with different allele frequencies and outcome distributions, may violate this second condition as well. In the Mendelian randomization context, confounding may exist if the subgroups (these often correspond to ethnic groups) are associated to both Z and Y [In order to use Mendelian randomization to infer causality in observational epidemiology, numerous conditions need to be fulfilled ,55–57. A X and U  wrote thr et al. . At the rest, Y) . However variant . Similar Z and Y .i.e., Z having multiple effects, which do not pass through X). This is however only a problem if the other functions of Z are associated to Y [C677T MTHFR variant has been associated with fetal viability [i.e., the effect of the paternally transmitted allele should be the same as the effect of the maternally transmitted allele).Also, there should be no pleiotropy,  only explain a very small proportion of variance of the phenotype of interest (X). This implies that very large sample sizes are usually needed  to reach sufficient power. Second, for many associations of interest, it is not possible to find an appropriate instrumental variable. However, as many more instruments are being discovered, the prospects are improving. Third, the success of this method heavily rests on the existence of allelic homogeneity, i.e., a common causal allele is shared by many individuals. Fourth, whereas analytic methods have been described for continuous outcomes, it is unclear to what extent these methods also apply to dichotomous outcomes. Considering the clear interest for epidemiologists to apply this concept for dichotomous outcomes such as diseases, it would be important, and even urgent, to clarify the issues on the validity of the instrumental variable approach in this context. More methodological development is needed before the instrumental variable approach can be confidently used for binary outcomes.The Mendelian randomization approach in observational epidemiology is a valuable tool that has taken a new dimension in the post-genomic era and is being used increasingly. This approach conceptually relies on an instrumental variable approach. There have been some successes of the Mendelian randomization approach to help unraveling causal relationships in observational epidemiology. Examples are the recently published evidence for the causal role of body mass index on blood pressure  or accum


Clinical studies have confirmed that renal oncocytoma (RO) is a benign neoplasm with excellent prognosis. In diagnostically challenging cases of renal oncocytic epithelial neoplasms, fluorescent in-situ hybridization (FISH) is increasingly being used and its ability to distinguish RO from chromophobe renal cell carcinoma (ChRCC) has been documented. In this study, we evaluated the differential diagnostic contribution of FISH in cases of RO.Clinicopathologic data and glass slides from 73 patients with RO were reviewed; 20 cases of ChRCC were included for comparison. FISH analysis of formalin-fixed, paraffin-embedded sections was performed using centromeric probes for chromosomes 1, 2, 7 and 17. FISH analysis revealed ROs had frequent loss of signal for chromosome 1 (56%) and 17 (44%). Tumors with more than one loss were common (41%) and 10% cases showed loss of all chromosomes examined. A total of 18% cases did not show any abnormality.Our study shows that chromosomal abnormalities in both ROs and ChRCCs are common with frequent loss of chromosomes 1 and 17. No association was found between overall patient survival and the extent of chromosomal abnormalities. FISH results, even those showing significant chromosomal abnormalities, should not alter the primarily morphology-based diagnosis of RO. Renal oncocytomas (ROs) comprise approximately 3-7% of renal epithelial neoplasms -3. CommoCytogenetic studies of RO revealed genetic heterogeneity of these neoplasms; three distinct subsets were identified: 1) translocation between 11q13 and other chromosomes, 2) loss of 1/1p followed by loss of chromosome Y or 14 and 3) non-recurrent or no detectable aberrations . MoleculBased on the current data on genetic abnormalities occurring in ROs, we have employed an abbreviated FISH panel for chromosomes 1, 2, 7 and 17 in our institution that would aid in their differential diagnosis. In this study, we present our experience with this FISH panel and its contribution in the differential diagnosis along with survival data of 73 patients with RO.The pathology records of the University of Pittsburgh were reviewed to find all patients who underwent primary nephrectomy for renal oncocytoma in the period of 25 years (1981 - 2006). Seventy-three cases of renal oncocytoma were selected from the UPMC archives using conventional histologic examination by two pathologists. For each patient, the data from the final surgical pathology report as well as from the clinical records were retrieved. In addition, 20 cases of ChRCC (11 cases of classic and 9 cases of eosinophilic variant) were included for comparison. Since the previously published FISH results did not show significant difference in observed abnormalities between the classic and eosinophilic variant of ChRCC , the twoFormalin-fixed paraffin-embedded sections were mounted and serially sectioned at 5-mm intervals. H&E section was used by the pathologist to determine the area of the tissue to be targeted for analysis. FISH slides were deparaffinized in xylene twice for 10 minutes, dehydrated twice with 100% ethanol and then pretreated using the Vysis Paraffin Pretreatment Kit. Slides were digested for 18 minutes in protease solution (0.5 mg/ml) at 37°C. FISH was performed using CEP1, CEP2, CEP7 and CEP17 centromere probes . The target slide was denatured at 75°C for 5 minutes and dehydrated in 70%, 85%, 100% ethanol. Slides were incubated with probe overnight at 42°C in a humidified chamber. Post-hybridization washes were performed using 0.4× SSC/0.3% Igepal (Sigma) at 72°C for 2 minutes, followed by a room temperature 2 × SSC/0.1% Igepal wash for 30 seconds. Slides were air-dried in the dark and counterstained with DAPI . Analysis was performed using a Nikon Optiphot-2  and Quips Genetic Workstation equipped with Chroma Technology 83000 filter set with single band excitors for Texas Red/Rhodamine, FITC, DAPI (UV 360 nm) . Only individual and well delineated cells were scored, overlapping cells were excluded from the analysis. At least 60 cells were analyzed in the targeted region. Signal loss was considered significant if present in more than 30% of cells, gain if present in more than 20% of cells examined.In the RO group, the study population included 51 males and 22 females  with an average age of 67 (range 29 - 83) years. A total of 32 patients 44%) underwent partial nephrectomy (nephron sparing surgery), the remaining 41 patients (56%) radical nephrectomy with or without ipsilateral adrenalectomy. The mean tumor size was 4.0 cm (range from 1 - 21 cm). Small vessel invasion was present in 4 (5.5%) cases, and invasion into perinephric adipose tissue in 5 (6.8%) cases. None of the patients had evidence of metastatic disease at diagnosis. After a mean follow-up of 64 months (range from 1 to 293 months), a total of 46 (63%) of patients were alive with no evidence of disease; 14 (19%) had died of other causes and no follow up was available for 13 (18%) patients. None of the patients had disease recurrence nor developed metastases. The control ChRCC group consisted of 11 males and 9 females male to female ratio 1.2:1) with a mean age of 64 (range 35 - 88) years (Table 1). Average tumor size was 6.1 cm (range 1 - 14.5 cm). A total of 8 patients (40%) were treated with partial nephrectomy, 12 (60%) with radical nephrectomy. Small vessel invasion was present in 3 (15%) cases, and invasion into perinephric adipose tissue in 2 (10%) cases; one patient had tumor grossly extending into the renal vein. After a mean follow up of 60 months (range from 25 - 87 months), one patient with a tumor extending into the perinephric adipose tissue developed local recurrence 25 months after a radical nephrectomy. Similarly to the renal oncocytoma group, none of the patients with available follow-up died of disease or developed metastases.Characteristic histological features of both RO and ChRCC were observed Figures  and 2. RIn 32 cases, the initial diagnosis of renal oncocytoma was based on histology alone (44%); a total of 41 (56%) cases had some ancillary studies, such as electron microscopy , DNA ploidy analysis , FISH  or histochemistry for Hale's colloidal iron . A total of 68 cases (93%) were initially diagnosed as oncocytoma; however, 5 cases (7%) were diagnosed using descriptive terms such as "atypical oncocytic neoplasm" or "renal cell carcinoma of oncocytic type (oncocytoma)". In contrast, all of the chromophobe renal cell carcinomas were initially worked-up using ancillary studies such as Hale's colloidal iron histochemical stain  or FISH .The FISH results are summarized in Table 2 and Figure ChRCCs consistently showed multiple chromosomal abnormalities, and 6/20 30%) of cases were abnormal in all chromosomes examined  was observed in papillary renal cell carcinoma ,25 that In our current study, the incidence of chromosomal abnormalities in ROs  was significantly higher than in the previous reports ,12,18. BA total of 3/73 RO cases showed loss of chromosome 1 and gain of chromosome 7 and/or 17; morphologically, 2 cases displayed characteristic alternating nested and organoid growth patterns; the third case had predominantly tubulocystic arrangement of typical oncocytes. All three were initially diagnosed as RO without any ancillary studies. Gains of chromosome 7 and/or 17 without any accompanying chromosomal loss was observed in 4/73 cases; histologic review confirmed the initial diagnosis of RO in all four cases; however, only some of the original histologic sections were available for review in 3 cases precluding a definitive diagnosis. RO cases with extracapsular or vascular space invasion usually showed multiple chromosomal losses or gains; however, one of the cases with perirenal soft tissue extension was normal by FISH. Long term follow-up of patients included in our study confirmed the excellent prognosis of RO as none of the patients died of disease, developed metastases or local recurrence. No association was found between overall patient survival and the extent of chromosomal abnormalities detected by FISH.The incidence of chromosomal abnormalities in ChRCC was in agreement with previous studies ,15,16; 1Our FISH study of ROs, which is largest to date, was based on a series of well-defined oncocytomas diagnosed according to the current morphologic WHO classification criteria . Our resThe authors declare that they have no competing interests.MD carried out the research studies, participated in the drafting of the manuscript. KMC, CRS and TLM carried out the FISH studies. MBA carried out the immunohistochemical studies. RD and SIB participated in the design of the study. AVP conceived of the study, helped draft the manuscript and participated in its design and coordination. All authors read and approved the final manuscript.


Inflammatory Bowel Diseases (IBD) remain significant health problems in the US and worldwide. IBD is most often associated with eastern European ancestry, and is less frequently reported in other populations of African origin e.g. African Americans ('AAs'). Whether AAs represent an important population with IBD in the US remains unclear since few studies have investigated IBD in communities with a majority representation of AA patients. The Louisiana State University Health Sciences Center in Shreveport (LSUHSC-S) is a tertiary care medical center, with a patient base composed of 58% AA and 39% Caucasian (W), ideal for evaluating racial (AA vs. W) as well and gender (M vs. F) influences on IBD.In this retrospective study, we evaluated 951 visits to LSUHSC-S for IBD (between 2000 to 2008) using non-identified patient information based on ICD-9 medical record coding (Crohn's disease 'CD'-555.0- 555.9 and ulcerative colitis 'UC'-556.0-556.9).Overall, there were more cases of CD seen than UC. UC and CD affected similar ratios of AA and Caucasian males (M) and females (F) with a rank order of WF > WM > AAF > AAM. Interestingly, in CD, we found that annual visits per person was the highest in AA M (10.7 ± 1.7); significantly higher (* -p < 0.05) than in WM (6.3 ± 1.0). Further, in CD, the female to male (F: M) ratio in AA was significantly higher (*- p < 0.05) (1.9 ± 0.2) than in Caucasians (F:M = 1.3 ± 0.1) suggesting a female dominance in AACD; no differences were seen in UC F: M ratios.Although Caucasians still represent the greatest fraction of IBD (~64%), AAs with IBD made up >1/3 (36.4%) of annual IBD cases from 2000-2008 at LSUHSC-S. Further studies on genetic and environments risks for IBD risk in AAs are needed to understand differences in presentation and progression in AAs and other 'non-traditional' populations. Saccharomyces cerevisiae (ASCA) and IBD-specific p-ANCA markers) [Crohn's disease (CD) and ulcerative colitis (UC), the major forms of inflammatory bowel disease (IBD), are characterized clinically by diarrhea, weight loss and fever as well as endoscopic, radiologic, histopathologic findings and biochemical markers (e.g. perinuclear anti-neutrophil cytoplasm (p-ANCA), anti-markers) . The devmarkers) -5. Thesemarkers) -10. IBD markers) ,12.In a study of central-African (Ghana) black patients  CD was vIt is possible that clinical features of IBD may differ in AAs, leading to misdiagnosis and under-reporting of 'minority' IBD . EidelweIncongruities about the incidence of IBD in AA populations may thus reflect low sampling, geographical influences, variations in disease presentation, environmental and socioeconomic factors that confound diagnosis and ultimately treatment. Gender-based differences in IBD have also been reported ; the F: 'Arklatex''). The racial composition of Shreveport has been >50% AA since 2002 [One important limitation in this type of study is adequate sampling, in this case, ideally selecting populations in which AA patients at least match, or even exceed that of Caucasians. Our study carried out at the LSU Health Sciences Center in Shreveport (in accordance with HIPAA regulations regarding privacy) should approximate local ethnic profiles in Shreveport,/Arkansas-Louisiana-Texas region  from information medical record codes for CD (555.0 - 555.9) and UC (556.0 - 556.9). For every year in the study, individual non-identified patients were evaluated for self-identified race, gender and ICD-9 coding, . Ages of individual patients at the time of annual visits were also recorded. Cumulative cases of CD or UC visits per each group were also counted (independent of the number of patients). We then determined: 1) the annual number of patients for each code, 2) the number of hospital visits per group, 3) the age at visit for individual racial and 4) gender populations at the LSUHSC in Shreveport, LA, (between 2000 and 2008). Hospital visits per individual were calculated as the total number of hospital visits (separated by race and gender) for each specific condition (CD or UC) and dividing this number by the number of cases for each condition. One-way ANOVA analysis was used to determine statistical significance with Bonferroni Multiple Comparisons post-testing. Statistical significance between pairs of data was determined using unpaired student's t-test. Nonlinear regression was used to calculate correlation and determine fit for trend lines in graphs. Because Hispanic, Asian, and Native American populations constitute <1% of total case reports for ICD-9 codes, these groups were omitted from analysis; we compared Caucasian and African-American populations with a further sub-grouping for gender. Co-morbid conditions with CD and UC, including diabetes (ICD-9 code 250.0), multiple sclerosis (ICD-9 code 340), and arthritis, were also investigated.This study was reviewed and approved by the LSUHSC-Shreveport Human Research Protection Program/Institutional Review Board (IRB). We used non-identified ICD-9 codes (Data were collected on patients with CD and UC at LSUHSC-S between 2000-8. The compiled data set included: 1) the total number of annual visits, 2) the annual number of cases, 3) the age at visit for each disease and 4) these factors separated both by genders and by race at LSUHSC-S. Over the past 9 years, 665 patients with CD and 286 with UC treated at LSUHSC-S were included in this study. Of CD patients, 428 (64.36%) were Caucasian, and 237 (35.64%) were African-American. When the CD population was subdivided by race and gender, 240 (36.09%) were Caucasian Fs, 188 (28.27%) were Caucasian M, 150 (22.56%) were AA F, and 87 (13.08%) were AA M. Caucasians represented 63.99% (183) of UC patients, while African-Americans comprised 36.01% (103) in this study. When UC patients were subdivided by race and gender, 38.46% (110) were Caucasian F, 25.52% (73) were Caucasian M, 23.08% (66) were AA F, and 12.94% (37) were AA M  for both CD and UC was also studied. These data only reflect the average patient age at hospital visit, and do not infer the age of initial diagnosis. ; . There was no statistical significance in average age amongst the population groups studied within each respective disease. When comparing CD to UC, at LSUHSC-Shreveport we did find that the average age at time of visit for CD to be significantly lower than for UC patients amongst Caucasian females  and Caucasian M .The annual number of cases per race/gender group was also compared for each condition. In both CD and UC, the average annual number of cases was highest amongst Caucasian F followed by Caucasian M followed by AA F, and finally the lowest number of cases was seen amongst AA M. When comparing this data in CD, Caucasian females had significantly more cases per year than AA females . This was also seen when comparing Caucasian M to AAM  Fig. . In UC, At LSUHSC-Shreveport, IBD affected more Caucasian (64%) than AA individuals (36%). We also compared the ratios of gender and race for both CD and UC. When comparing this ratio (W: B), men were affected more than women in CD  Fig. . In UC wIBD  has long been considered as a group of diseases that affect primarily Caucasian populations ,27, an iEidelwein et al.  also sugCaspase recruitment domain-15 'CARD15' may dysregulate intestinal epithelial apoptosis and barrier [Several genes linked with development of IBD, have been compared in AA and Caucasian populations. In IBD, polymorphisms in  barrier . Kugatha barrier  correlat barrier . Additio barrier .Nucleotide-binding oligomerization domain containing 2). 3 polymorphisms in the NOD2/CARD15 gene  (16q12) are risk factors for CD [Another risk factor for CD is the NOD2 gene  and may represent a significant 'surrogate' marker of disease, . In our study between 2000-2008, the Caucasian: African-American patient ratio was 0.67:1. At the same time we found that more than 1/3 of IBD cases (35.64%) were seen in African-Americans, vs. 64.36% of IBD cases who were Caucasian (W). The Caucasian: African American IBD ratio is 1.8:1. Based on the ratios from our center, Caucasians were 2.68 times more likely to develop a form of IBD. We found a greater proportion of female cases in both CD and in UC with a male: female ratio of  in CD and a male: female ratio of  in UC. This ratio is reversed from that reported by Kugathasan et al.  who repoAmong UC patients at LSUHSC, 286 patients were seen over 9 years. Of these, 63.99% n = 183) were Caucasian; of these, 38.46% (n = 110) were female and 73 (25.52%) were male. African-Americans comprised 36.01% (n = 103) of UC patients; of these 23.08% (n = 66) were female and 12.94% (n = 37) were male .nd highest rate of hospital visits  for CD, (not significantly different vs. WF). While preliminary, hospital visits may represent a potential 'surrogate' marker of disease activity requiring additional study.Although the number of hospital visits at the LSUHSC-Shreveport for CD and UC have been increasing annually in Shreveport since 2000, total visits have also increased, and the fraction of cases for each condition has not changed significantly. Our results among these groups show that AA M had more hospital visits  for CD than other groups; this was not found when analyzing UC visits Fig. . AA femaIn summary, we provide racial, gender and age statistics of IBD at the LSUHSC in Shreveport over the past 9 years. Our institution serves more AAs than any other racial group and provides an important site for opportunity to study IBD in African Americans. While Caucasians represented the most cases of IBD, AAs represented 36% of IBD cases with the highest annual visits among AA males. Classical assumptions about the prevalence and course of IBD in AAs may underlie the failure to suspect, diagnose, and treat IBD in this population.The authors declare that they have no competing interests.HV, performed data compilation and analysis, graphic and manuscript preparation. KS performed data compilation and statistical analysis, graphics and manuscript preparation. AS, performed data compilation and analysis, and worked on introduction and discussion. ES performed statistical analysis, and manuscript introduction and discussion. SW performed final data compilation, bibliographic, graphics and worked on manuscript introduction and discussion. AP coordinated and collected de-identified data sets from hospital records. GVC helped develop the concept for the paper, supervised statistical analysis, assisted in writing the introduction and discussion. KM coordinated fellow participation, assisted in writing the introduction for the manuscript. KB performed data analysis and statistics and contributed to introduction. EZ contributed to introduction and conclusion sections. MB assisted with data analysis, graphics and introduction. WS assisted in statistical analysis, graphics and in writing the manuscript. JMM assisted with data analysis, and discussion. BD initiated data collection and analysis (with AP). PJ assisted in developing the overall concept, interpreted data, wrote introduction, discussion and conclusions. AM interpreted data and assisted in writing conclusions. JSA developed the overall concept, supervised collaborators and wrote introduction, discussion and conclusions. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/10/104/prepub