These profiles are being adapted for human epidemiology studies, given the importance of energy balance in human disease.Biomarker-based assessments of biological samples are widespread in clinical, pre-clinical, and epidemiological investigations. We previously developed serum metabolomic profiles assessed by HPLC-separations coupled with coulometric array detection that can accurately identify Human plasma samples were biochemically analyzed using HPLC separations coupled with coulometric electrode array detection.We identified these markers/metabolites in human plasma, and then used them to determine which human samples represent blinded duplicates with 100% accuracy (N = 30 of 30). At least 47 of 61 metabolites tested were sufficiently stable for use even after 48 hours of exposure to shipping conditions. Stability of some metabolites differed between individuals , suggesting the influence of some biological factors on parameters normally considered as analytical.Overall analytical precision  and total between-person variation  appear well suited to enable use of metabolomics markers in human clinical trials and epidemiological studies, including studies of the effect of caloric intake and balance on long-term cancer risk. After tobacco, over-nutrition is, arguably, the major cause of excess morbidity in developed countries, affecting a broad spectrum of diseases including cancer, cardio-/cerebrovascular disease, and type II diabetes. This association may be seen in both broad demographic groups, such as the American Cancer Society study group  and in mMetabolomics technology ,17 offerOur metabolomics approach to clinical and epidemiological questions is distinct from, and complementary to, both direct targeted analysis  and global profiling. Specifically, we focused initially on an animal model that displays the physiological benefits associated with nutritionally replete, lower-energy diets-21,36,37We have previously completed proof of principle studies showing that we can identify serum metabolites that differ between AL-fed rats and rats undergoing CR, confirmAs we move from studies of rat sera to studies of human plasma, however, many of the potential sources of error become both qualitatively different and quantitatively more complex. Here we address two of these issues: (i) our ability to measure these analytes reproducibly in banked human plasma and (ii) the need to assess the stability of these markers under different, realistic, and "worst case" shipping conditions. We note that, for the purposes of this report, we define this "worst case" in the context of the specific samples we expect to test in the future, which are drawn from the NHS. These samples are handled exactly as are the samples in the current report, and no sample is used that has been held >48 hours in shipping conditions.Two sets of plasma samples that were collected into sodium heparin-containing tubes for use as analytical controls were examined in this study. Approximately 75% of the blood samples were drawn at least 8 hours after the last meal. In our study, all analysis of samples was conducted in a blinded fashion. Set 1 was comprised of duplicate (split) samples from 12 women who were participants in the NHS and triplicates (splits) of two pooled plasma samples. The latter samples were pools made up of multiple units of fresh frozen plasma obtained from a local hospital and used routinely to evaluate laboratory reproducibility. Set 2 consisted of three (split) samples from each of 10 individuals as well as duplicates of two pooled plasma samples as described above. These 10 adults were healthy men and women recruited locally who responded to a flyer requesting volunteers to provide blood samples for pilot studies. Of the three blood samples from each individual in Set 2, the first was processed (see below) immediately after acquisition, the second was stored as whole blood in a refrigerator for 24 hours, and the third was stored for 48 hours; these latter conditions mimicked typical overnight shipping conditions in many cohort studies. Processing consisted of centrifugation of whole blood samples at 1530 g for 20 min at 4°C, after which the plasma was removed and aliquots placed into cryotubes. These cryotubes containing plasma samples were frozen and maintained in the vapor phase of a liquid nitrogen freezer at <130°C. All nitrogen freezers were alarmed and monitored continuously. Samples were shipped from The Channing Laboratory to Burke Medical Research Institute by overnight courier on dry ice. Further details on this approach have been previously published[IRB approval was obtained from Partners Human Research Committee and the IRB at Burke Medical Research Institute.Metabolite extraction, separation, detection, and identification were conducted as previously described. ,38,40-45Chromatographic separation and electrochemical detection were performed using HPLC coupled with an electrochemical array detector (HPLC-ECD), as previously described,45. The The gradient essentially displays an increase in hydrophobicity from that of ascorbate to that of tocopherol. Detection of metabolites was accomplished with a 16-channel coulometric array detector with potentials incremented in 60 mV steps (0–900 mV). All HPLC-ECD system functions were controlled by CoulArray software; biomarkers were identified and quantitated using CEAS-5.12 software. The metabolite concentration in individual human plasma samples was assessed and reported relative to that of metabolites in the "model" pool, in which the concentration of all markers was set at 100. The metabolic profile of the human pool studied in this report includes up to 66 markers, metabolites that represent a subset of the ~90 metabolites that we previously identified in sera of rats fed either AL or calorie restricted diets ,18,19,21Most of the metabolites studied here are identified by virtue of their position in the array (retention time) and their relative reactivity across the array (dominant and subdominant channel). Examples of metabolites that can be assessed via Coularray-based technology include some amino acids , the majority of the tryptophan and tyrosine catabolic pathways, indoles, purines, antioxidants  and redox damage products .Pearson correlation matrices were calculated in NCSS 97 with pairwise deletion for missing data. Means and errors, data simulations, and chi-squared analysis were conducted/determined in Microsoft Excel 2002. Paired t-tests were conducted in Statview. Principal components analysis was conducted using SIMCA P10.5 . A single metabolite value, present at apparently 100-fold higher levels than any cognate metabolite in different samples, was excluded as an outlier.Our previous analytical validation studies focused on identifying the sources of potential analytical error in our analyses, including methods of sample acquisition and preparation, handling, transportation, and storage, as well as the influence(s) of total series size, complexity of the organic matrix, and aspects of experimental design,15,47,48The reproducibility (precision) of metabolite measurements was addressed by blinded analysis of split samples. In the framework of a reproducibility study, aspects of the analytical platform such as the delivery of a sample to the analytical laboratory and the completion of sample analysis , chromatographic separation and electrochemical detection, and peak identification and quantitation were considered.To test our ability to analyze human plasma, we received 30 blinded samples, each of which was present in duplicate or triplicate . At the initial step, we looked for previously identified CR markers in the samples and identified 66 metabolites that clearly were present in serum/plasma from both species and were analytically suitable without further optimization.Of the 23 remaining markers in our standard rat profile, 12 were not present in the plasma sample , and 11 represented unclear assignments and were not studied further in this report.analytical self-similarity represents the first stage in validating the analytical platform for future use in human studies. In all 30 cases, cognate duplicates/triplicates were immediately apparent  were scored for the 66 markers . The resulting data were then used to estimate the analytical accuracy that could be obtained from the analysis of banked human plasma , 46 (~75%) showed variation consistent with normal analytical variation over this time frame  enabled us to assign 29 of 34 samples to the individual from whom the sample was derived  had essentially equivalent levels of metabolites . This result was observed for most markers when they were compared between most individuals in our data set Figure . Samples-10.To assess the statistical validity of this observation of apparent inter-personal differences in compound stability, we compared all metabolite changes by paired t-test . Twenty of 45 comparisons had p < 0.05 at 24 hours, and 22 of 45 had p < 0.05 at 24 hours. To assess the likelihood of this result occurring by chance, we modeled changes in metabolite levels assuming that all changes were random. Of 100 comparisons, 6 had p values < 0.05 . Chi-squared analysis of the comparison resulted in p values of < 10The central finding of this paper is that the metabolites that we have previously used to distinguish caloric intake in rats can be analyzed in human plasma with good analytical precision (median CV of 9–12%) and have high inter-sample variability (median CV of 50–70%). This combination, similar to results obtained from rat studies,  suggestsTwo important caveats follow from our experimental design: (i) the estimate of total inter-sample variability includes both the analytical variability and the biological variability, although the study's overall analytical precision suggests that the variability is primarily biological in origin; and (ii) we cannot distinguish the components of biological variability that derive from sample-to-sample within-person variability versus long-term between-person variability – the latter of which is critical for our planned investigations. Work on this latter question, the relative biological variability between different people as compared to the variability within a temporal series of samples from a given person, is the next logical step.In general, biomarker validation studies require demonstrating validity in three broadly defined stages, in which the following concerns are addressed: (i) analytical issues ,47,48;  must precede stage (ii) and that stage (i) is essentially independent of stage (ii). In part, this logical construction simply states that we must be able to measure the concentration of an analyte, and understand the limits of that measurement, before we can usefully examine differences in that analyte between two or more conditions of interest. The above logical construction further implies that our ability to measure a given analyte and the basic analytical properties of that analyte are expected to be unaffected by its source – that is, the person from whom the sample is derived .Our results appear to provide empirical evidence supporting a noteworthy exception to this logical, but ultimately theoretical argument. These data, and the interpretation of these data, are dependent on the extent to which sample handling in our experiment was sufficiently controlled to enable other influences to be excluded. In support of the idea that we met these conditions, the differences observed are primarily in the 48 hour samples, whereas we would expect random distribution for most possible analytical problems . Further support for our ability to generally fulfill the goal of appropriate sample handling is provided by evidence of high correlations between the levels of corresponding metabolites in paired samples  rigorously defined, but would be difficult to address in humans due to both biological and analytical noise. Understanding analytical noise is thus one step toward enabling study of mechanistic hypotheses in humans.In conclusion, it is worth recalling that the development and validation of biomarkers of nutritional status for use in human studies has a long history. Despite this history, relatively few useful markers – other than direct intake markers such as carotenoids in blood, double-labeled water, and urinary nitrogen – have been identified and are in widespread use for dietary status assessment ,58-60. ACR: Calorie-restricted;HPLC-ECD: HPLC coupled with an electrochemical array detector;NHS: Nurses' Health Study.Dr. Matson was a founder and former director of ESA, Inc, which makes the analytical equipment used in this study, but he no longer has a financial interest in the company. Both Drs. Kristal and Matson consult for and have equity interest in Metabolon, a metabolomics company, but the work appears unrelated to Metabolon.Dr. Kristal has equity and potential royalties from licensed intellectual property to Metabolon , but this patent is also not directly related to the work presented here.Drs Kristal and Matson were joint inventors on a patent on biomarkers of caloric restriction, which is unlicensed. Dr. Kristal's interest is again overseen by Weill Medical College of Cornell University , Dr. Matson's interest is wholly owned by ESA, inc.YIS contributed to the analytical design of this work, conducted and analyzed all HPLC analysis, and drafted the manuscript, WRM contributed to the overall/general conception and design of the HPLC analysis, WCW and SEH contributed to the conception and design of the human sample studies in this work, BSK conducted the statistical and informatics analysis and drafted the manuscript. All authors contributed to the overall design of the larger project of which this work is a part, and all authors were involved in editing and approving the manuscriptThe pre-publication history for this paper can be accessed here:


Multi-detector computed tomography angiography (MDCTA)of the coronary arteries after stenting has been evaluated in multiple studies.The purpose of this study was to perform a structured review and meta-analysis of the diagnostic performance of MDCTA for the detection of in-stent restenosis in the coronary arteries.A Pubmed and manual search of the literature on in-stent restenosis (ISR) detected on MDCTA compared with conventional coronary angiography (CA) was performed. Bivariate summary receiver operating curve (SROC) analysis, with calculation of summary estimates was done on a stent and patient basis. In addition, the influence of study characteristics on diagnostic performance and number of non-assessable segments (NAP) was investigated with logistic meta-regression.Fourteen studies were included. On a stent basis, Pooled sensitivity and specificity were 0.82(0.72–0.89) and 0.91 (0.83–0.96). Pooled negative likelihood ratio and positive likelihood ratio were 0.20 (0.13–0.32) and 9.34 (4.68–18.62) respectively. The exclusion of non-assessable stents and the strut thickness of the stents had an influence on the diagnostic performance. The proportion of non-assessable stents was influenced by the number of detectors, stent diameter, strut thickness and the use of an edge-enhancing kernel.The sensitivity of MDTCA for the detection of in-stent stenosis is insufficient to use this test to select patients for further invasive testing as with this strategy around 20% of the patients with in-stent stenosis would be missed. Further improvement of scanner technology is needed before it can be recommended as a triage instrument in practice. In addition, the number of non-assessable stents is also high. Coronary artery disease (CAD) remains one of the leading causes of death and morbidity in the Western world. Each year one million patients are treated percutaneously with stent implantation . UnfortuInvasive coronary angiography (CA) is the standard of reference for the evaluation of stent patency and the exclusion of in-stent restenosis (ISR). Although it has a small risk of complications it is an invasive procedure with significant costs -6. A nonMulti-detector computed tomography angiography (MDCTA) of the coronary tree has been evaluated in multiple studies, to assess the patency of the lumen after stent implantation -20. All We tried to frame the question starting from the potential future use of MDCTA in evaluation of stent patency. A potential use that is probably reasonable is using MDCTA as a triage modality on the result of which a decision can be made whether the patient needs invasive angiography . This caTo search for original articles, a structured search of the PUBMED database from January 1998 to March 2007 was performed using the previously described PICO search strategy , by threThe study parameters were extracted first independently and subsequently by consensus if a disagreement existed between the observers concerning the numeric value of a parameter . The absolute numbers of FN, FP, TP, TN were retrieved or calculated. The numbers were calculated with Bayes theorem if only values for sensitivity, specificity, and predictive values were reported. This was done on a per stent basis and per patient basis.MDCTA was considered true-positive per stent if an in-stent restenosis (≥ 50% diameter stenosis) was found on MDCTA and confirmed on CA. MDCTA was considered true-negative if in-stent restenosis was correctly ruled out. MDCTA was considered false-negative if no in-stent restenosis was found on MDCTA and one was found on CA. MDCTA was considered false-positive if it revealed an in-stent restenosis and CA showed no in-stent restenosis. For the per patient analysis a patient was classified in one of these categories according to the authors decision.All values are expressed as mean value with 95% confidence intervals (95%CI) unless otherwise specified.Interobserver agreement for study selection was evaluated with Cohen's Kappa test in which a value higher than 0.8 is considered to imply very good to excellent agreement. The different rounds of study selection were evaluated.The analysis was performed on a per stent basis, as most studies focused on this level of information. We also did a evaluation on a per patient basis.2 statistic, and is a derivative of Cochran's Q [We evaluated potential heterogeneity and inconsistency between publications expressed with the Higgins and Thompson index  which cahran's Q ,30. CochPublication bias was assessed according to the method introduced by Deeks . This meWe performed a summary receiver characteristic operating curve (SROC) analysis tailored to meta-analysis of diagnostic accuracy that takes into account any possible correlation between sensitivity and specificity in addition to within-study variation (precision) and between study variation (random effects approach). We used the bivariate meta regression approach since recent work has shown that the standard approach ,35 can pThe PUBMED search for original articles resulted in 485 articles. 449 articles were excluded on the basis of their title or abstract with 36 remaining for further evaluation. From these 36 articles, 11 were finally included in the meta-analysis -12,14-20The final group of studies consisted of 4 studies on 64-detector CT, 1 on 40-detector CT, 1 on a combination of 64- and 16-detector CT, and 8 on 16-detector CT. 24 studies were excluded. Number and reasons for exclusion are found in Table Interobserver agreement (Cohen's Kappa) for the selection of articles between the three readers was respectively 0.75, 0.87 and 1.0 for the different search rounds.Important study characteristics are displayed in Table A total of 1039 stents were analyzed. In the per patient analysis 400 patients were analysed.Raw data from the included studies are displayed in Table 2, 91.42%, 79.51%, respectively).Substantial inconsistency between studies was found when calculating the pooled SE and SP on a stent basis with the bivariate model , Intercept 3.10 , Intercept 2.96 The pooled values for detecting significant ISR on a stent basis were as follows: Pooled sensitivity and specificity were 0.82 (0.72–0.89) and 0.91 0.83–0.96). Pooled negative likelihood ratio and positive likelihood ratio were 0.20 (0.13–0.32) and 9.34 (4.68–18.62) respectively. Values on a patient basis are found in figures –0.96. PoForest plots for pooled sensitivity, specificity, are graphed in figures Random effects SROC curve and conditional probability plots are displayed in figure The results of this study showed that pooled sensitivity and specificity on a stent basis were 0.82 (0.72–0.89) and 0.91 (0.83–0.96) which is moderate (sensitivity) to very good (specificity). Considerable inconsistency/heterogeneity was found between all included studies. There was no difference in the diagnostic performance between scanners with 16 or 64 detectors and the only variable that showed to have influence on diagnostic performance was the exclusion of non-assessable stents before analysis. The proportion of stents that was non assessable showed a very important variability, and was in one study as high as 0.46 and showed a pooled value of 0.11. Meta-regression on NAP showed that it was influenced by the number of detectors, stent diameter, strut thickness and the use of an edge-enhancing kernel. This is probably reflecting evolving stent technology, as will be stated later in this discussionPooled negative likelihood ratio and positive likelihood ratio were 0.20 (0.13–0.32) and 9.34 (4.68–18.62) respectively. For a test to be helpful in diagnosis, it is generally accepted that LR+ should be higher than 10 and LR- below 0.1 .These results underscore the fact that demonstration of ISR with MDCTA can be done but still suffers from some problems, and that non-invasive imaging of stents to rule out ISR (moderate sensitivity) remains a challenge, even after the addition of 16 to 64 detector MDCTA to the diagnostic armentarium. The sensitivity is probably not good enough to use the technique as a rule-out triage modality.On the basis of studies in vitro, a logical and intuitively attractive concept has been formulated on stent imaging with MDCTA ,22. ThicIn addition to these stent related factors, scan acquisition parameters (number of detectors and the use of an edge enhancing kernel) had an influence, also through the number of non-assessable segments.Although these principles suggest that stent imaging with MDCTA is not always straightforward and prone to failure in certain stents, the general trend in interventional cardiology to use stents with thinner struts and less dense material will probably obviate at least some of this problem. Also scanner technology is advancing quickly and some of the scanner related problems will probably also be obviated in the future.As said previously, non-invasive evaluation of coronary artery disease after stent placement is a clinical challenge, and CA is not recommended as a routine follow-up technique due to the associated risks and cost. Therefore, non-invasive follow-up of ISR and re-occurrence of myocardial ischemia has been investigated with a variety of techniques. ECG stress testing has been tested and in a recent overview, sensitivity and specificity was 54% and 70% respectively, which is clearly insufficient for a triage modality . MyocardWe have to acknowledge some limitations in this study.The rather small size of the individual cohorts and the methodology (examining very different stents and stent sizes in each study) resulted in a heterogenous group of stents that make it cumbersome to reliably pool the data. This makes it also harder to obtain reliable results with the technique of multivariable logistic meta-regression, due to the very high number of explanatory variables (different stents) involved.Some clear disadvantages of MDCTA are the relatively high radiation dose that goes with the examination, with average doses ranging from 10 to 20 mS , and theSince MDCTA technology and stent technology is advancing very rapidly, future studies should focus on the use of state-of the art equipment both in terms of scan methodology and in terms of the stents investigated. If ISR is studied in patients, rigorous methodology to group enough stents of the same size and type should be done, preferably in larger and important anatomical locations. This would probably be very helpful to predict which patients and stents are good candidates to be investigated with MDCTA, with a reasonable change of obtaining images that can be reliably interpreted.Pooled analysis of diagnostic performance of MDCTA of ISR shows that the technique is probably useful, but that more thorough and uniform investigation of modern stents with the latest equipment will probably be needed to shed more light on the clinical usefulness of this technique. The diagnostic performance is influenced by the method of reporting (exclusion of non-assessable stents). The proportion of non-assessable stents is influenced by the number of detectors, stent diameter, strut thickness and the use of an edge-enhancing kernel.The sensitivity of MDTCA for the detection of in-stent stenosis is insufficient to use this test to select patients for further invasive testing as with this strategy around 20% of the patients with in-stent stenosis would be missed. Further improvement of scanner technology is needed before it can be recommended as a triage instrument in practice. In addition, the number of non-assessable stents is also high.The authors declare that they have no competing interests.PKV carried out the literature search, statistical analysis and drafted the manuscript. ID carried out the literature search, statistical analysis and drafted the manuscript. OB, GS and EV participated in the study design and collection of data. WW conceived of the study and participated in its design and coordination. BAD participated in the statistical analysis and coordinated methodologic aspects. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Appendix 1 studies that were excluded according to reason for exclusion.Click here for file


Previous studies showed an association between Coagulation Factor XII 46C>T polymorphism and variation in FXII plasma levels, as 46C>T seems to affect the translation efficiency. Case-control studies in Spanish samples indicated that genotype T/T is an independent risk factor for venous thrombosis, ischemic stroke and acute coronary artery disease. In this study, we tried to reaffirm the importance of 46C>T in two samples from Spain and Tunisia.A Transmission Disequilibrium Test (TDT) based on 101 family trios from Barcelona with one offspring affected by ischemic heart disease and a classical case-control study based on 76 patients with IHD and 118 healthy individuals from North and Centre-South Tunisia were conducted. Subjects were genotyped for 46C>T and data were analyzed accordingly, revealing no association in any of the two samples .The results suggest that 46C>T is not a risk factor for ischemic heart disease in any of the two analyzed samples and therefore the polymorphism seems not to be a universal risk factor for cardiovascular diseases. The gene of coagulation Factor (F) XVII has been repeatedly analysed in many epidemiological studies on cardiovascular (CV) genetic risk with controversial results. It has been reported that FXII plasma levels exhibit a 67% of heritability and that these levels are strongly determined by F12 gene variants.A highlighted F12 polymorphism is the 46C>T transition. It seems that 46C>T affects the translation efficiency, leading to reduced FXII plasma levels.[Previous genetic association studies between 46C>T and CV risk showed that genotype T/T increases significantly the risk for venous thrombosis, ischemic stroke and acute coronary artery disease in a Spanish population. -7 The odIn this context, this study provides additional data about the allele frequency distribution of 46C>T in two new Mediterranean samples. These data are used in an attempt to confirm previous genetic association studies, by a double epidemiological design: a family-based analysis (Transmission Disequilibrium Test - TDT) applied in a Spanish sample and a classical case-control study applied in a Tunisian sample.A total of 101 nuclear families (n = 302) with one offspring  clinically diagnosed for ischemic heart disease (IHD) were analyzed, all from the area of Barcelona, Spain. A comprehensive description of the families, diagnosis criteria and DNA extraction methods has been reported elsewhere.[® SNP genotyping assay protocol of Applied Biosystems for a total volume of 5 μl per well. DNA amplification as well as fluorescence intensity measurements of the final reaction product and data collection were carried out with an ABI PRISM® 7900 HT Sequence Detection System.Polymorphism 46C>T was genotyped by Real-Time PCR, using the standard TaqManAllele frequencies in the Spanish IHD patients and their parents as well as in patients and controls from Tunisia were calculated directly. A TDT analysis was performed in the IHD families from Barcelona, using the FBAT v2.0.2c program. In the TFrequencies of the T allele in the Spanish and Tunisian samples are shown in Table Our study showed that the allele frequencies of the FXII 46C>T polymorphism are similar in the Spanish and Tunisian samples, in accordance with the Caucasian pattern. This suggests a stable presence of 46C>T in different Mediterranean regions. On the other hand, our results indicate the absence of association between 46C>T and CV disease in both samples.The similar allele frequency distributions between Spanish patients and parents and between Tunisian patients and controls provide a first rough estimate of the lack of association. Moreover, our Tunisian sample has enough power (>99%) to detect an odds ratio of 4.8, as reported elsewhere, reinforcing the consistency of the lack of association detected. As far aThe diverse results of previous studies regarding association between polymorphism 46C>T and CV disease could be explained, at least in part, by the differences in study design, the definition of inclusion and exclusion criteria and the number of participants. In our case, the low sample size in the TDT study and the lack of a matched case-control design impose some caution in the interpretation of our results.In short, our results provide indirect evidence that 46C>T is not a universal independent risk factor for CV diseases. What seems to be well established is that the T allele affects the variation of intermediate phenotypes (FXII plasma levels). If this is true, then the diversity of results in genetic association studies is hard to explain. This observation leads us to adopt the idea that the low levels of FXII are the consequence rather than the cause of the disease, as has been recently suggested. This ideThe authors declare that they have no competing interests.GA carried out the SNP genotyping, participated in the statistical analyses and wrote the original manuscript. EE gave important advice for the improvement of the manuscript. MGV carried out the Spanish sample preparation. RCT helped with the statistical analyses. RB carried out the DNA extraction from the Tunisian samples. PM participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


The aim of our study was to investigate the relationship between a 5-HT1A promoter polymorphism and the binding potential of another selective 5-HT1A receptor antagonist, [18F]MPPF, in healthy subjects.Previous Positron Emission Tomography (PET) studies of 5-HT18F]MPPF scan and were genotyped for both the C(-1019)G 5-HT1A promoter polymorphism and the triallelic serotonin transporter gene-linked polymorphic region. We used a simplified reference tissue model to generate parametric images of BPND. Whole brain Statistical Parametric Mapping and raphe nuclei region of interest analyses were performed to look for an association of [18F]MPPF BPND with the C(-1019)G 5-HT1A promoter polymorphism.Thirty-five volunteers, including 23 women, underwent an MPPF BPND between groups was observed, except for two women who were homozygote carriers for the G allele and showed greater binding potential compared to other age-matched women over the frontal and temporal neocortex. However, the biological relevance of this result remains uncertain due to the very small number of subjects with a G/G genotype. These findings were not modified by excluding individuals carrying the S/S genotype of the serotonin transporter gene-linked polymorphic region.Among the 35 subjects, 5-HT1A promoter polymorphism and [18F]MPPF binding in healthy subjects. However our data suggest that the small number of women homozygote for the G allele might have greater [18F]MPPF BPND relative to other individuals. This finding should be confirmed in a larger sample.We failed to observe an association between the C(-1019)G 5-HT All subjects gave their informed consent to participate in this study, which was approved by the local Ethics committee  in accordance with the Declaration of Helsinki and French regulations on Biomedical Research.A and LA/LA  amplification was performed according to the protocol originally described by Hong et al. , and enasly; see  for deta3 voxels and an anatomical volume that covered the whole brain. Magnetic resonance images (MRIs) were visually analysed by a neurologist to ensure that no brain lesion or malformation was present.All subjects also underwent a 3-D anatomical T1-weighted sequence on a 1.5-T Siemens Magnetom scanner . Data were acquired in the sagittal plane with 1 mm18F]MPPF was acquired with a CTI Exact HR+ scanner for 60 min after the injection of 186 ± 24 MBq of [18F]MPPF. Specific activity ranged between 37 and 111 GBq/μmol , according to consensus nomenclature [18F]MPPF studies MPPF BPND in the dorsal and median raphe nuclei using a region of interest (ROI) delineated on the average [18F]MPPF BPND image obtained from the normalized scans of our subjects by setting the threshold activity at 90% of the local maximum in the brainstem. The ROI was then displayed on the MRI template to verify its proper location in the peri-aqueducal grey matter of the brainstem. This ROI extended on the 13 consecutive slices displaying the raphe, with a total volume of 1400 mm3. For each subject, ROIs were then applied on normalized and smoothed parametric BPND images to extract a mean BPND value for this ROI. The mean BPND values of raphe were compared among C/C, C/G and G/G subgroups in the total population, as well as in women and men separately, using an ANOVA and SPSS (version 12). The global BPND values and age were considered as co-variables of no-interest. The significance threshold was set at p < 0.05.We further analysed [18F]MPPF BPND MPPF BPND was observed between the three genotypes of C(-1019)G 5-HT1A promoter polymorphism .When considering the whole sample of 35 subjects or the subgroup of 12 men, no significant difference in [18F]MPPF BPND in homozygous carriers for the G allele compared to C/C and C/G individuals, while no difference was observed between these two latter populations. Differences between G/G and C/C subjects were observed in the anterior portion of the left superior and middle frontal gyri (pcorrected = 0.042) as well as over the right and left orbitofrontal cortex (pcorrected = 0.001) (see table corrected < 0.001), the anterior aspect of the right middle and inferior temporal gyri (pcorrected = 0.004), the right orbitofrontal cortex (pcorrected = 0.014) and the right frontal dorsolateral cortex (pcorrected = 0.007) G 5-HT1A promoter polymorphism, with greater BPND for those with G/G compared to those with C/C and C/G (see table corrected = 0.021) G 5-HT1A promoter functional polymorphism and [18F]MPPF/5-HT1A receptor binding. This negative finding could reflect an unsatisfactory statistical power. However it should be stressed that the primary analysis upon which this conclusion is drawn contrasted 21 subjects with C/G genotype and 11 subjects with C/C genotype, in accordance with the general empiric consensus that neuroimaging studies that include groups of 12 subjects benefit from adequate statistical power. The 9 additional subjects with C/G genotype are likely to compensate the marginally reduced statistical power related to the 11 subjects in the C/C group. Overall, we believe that the statistical power of our main negative findings, though necessarily uncertain, is fully consistent with that used in the great majority of PET studies worldwide.In this PET study, no clear cut association was identified between C(-1019)G 5-HT18F]MPPF BPND in various frontal and temporal brain regions than those carrying at least one C allele. However, the significance of this latter finding, which was shown to be independent of the previously reported association between 5-HTTLPR polymorphism and [18F]MPPF BPND in women, remains uncertain, due to the small number of individuals with a G/G genotype. Indeed, only three of our 35 healthy subjects (9%), including two women, were homozygous for the G allele, a figure consistent with the 8-10% prevalence of this genotype in the general population. This small number of subjects carries the risk of spurious PET findings, despite the strong statistical significance of our SPM analysis. It must be stressed that there is no easy solution to this limitation. The number of controls who can be scanned in any given PET centre is constrained by ethical issues, providing little justification to study larger populations than those reported here. Conversely, mixing data from healthy controls with those from patients suffering from various psychiatric disorders, reported in previous studies [1A promoter polymorphism in a sufficiently large series of normal subjects in order to select an appropriate number of subjects homozygote for the G allele who will then undertake a [18F]MPPF PET study.The women homozygote carriers for the G allele appeared to demonstrate greater WAY-100635 PET studies have previously investigated this issue, and have reported discordant results [11C]WAY-100635 BP in carriers of at least one G allele compared to the C/C genotype, over the raphe nuclei, the amygdala, and the hippocampus [1A polymorphism and [11C]WAY-100635 BP [11C]WAY-100635 BP reported in women in the two mixed population studies [18F]MPPF BPND which association with 5-HTTLPR and C(-1019)G 5-HT1A polymorphisms were found exclusively in women [11C]WAY-100635 BP selectively in women WAY-100 635 and [18F]MPPF to 5-HT1A receptor is reported to significantly decline with age, especially in women [18F]MPPF compared to [11C]WAY-100 635. Specifically, [18F]MPPF exhibits a relatively low affinity for 5-HT1A receptors, comparable to that of endogenous 5-HT, and appears to selectively bind to externalized receptors [11C]WAY-100 635 demonstrates greater affinity and a non-selective binding to both internalized and externalized receptors [18F]MPPF more sensitive than [11C]WAY-100 635 to the extracellular concentration of 5-HT, as suggested in rodents [18F]MPPF BPND might increase as a result of decreased 5-HT release [18F]MPPF BPND observed in healthy women homozygote carriers for the G allele, if confirmed, could reflect the downstream consequences of an increased expression of 5-HT1A autoreceptors in the raphe nuclei, i.e. a decreased release of 5-HT as well as a subsequent increased expression of post-synaptic 5-HT1A receptors. However, the lack of clear-cut changes in [18F]MPPF BPND over the dorsal and median raphe nuclei does not support such a mechanism.Other important methodological issues need to be considered when interpreting available PET data, including age of subjects, data analysis, and the molecular characteristics of ligands. Binding of MPPF BPND was not due to an imbalanced distribution of 5-HTTLPR alleles among the different groups considered. Interestingly, while both polymorphisms are associated with common features regarding [18F]MPPF BPND (same gender specificity and greater [18F]MPPF BPND in subjects with supposedly lower extracellular concentration of 5-HT), these were observed in distinct brain regions. Whereas differences in [18F]MPPF BPND associated with 5-HTTLPR polymorphism were previously reported predominately over paralimbic regions  [18F]MPPF BPND associated with C(-1019)G 5-HT1A promoter polymorphism, was primarily localised to the frontal and temporal neocortex in this study. These distinctive patterns might reflect the involvement of specific serotoninergic projection pathways.An original aspect of our study was to take into account both 5-HTTLPR and C(-1019)G 5-HTngulate) , [18F]MP1A promoter polymorphism and regional distribution of [18F]MPPF BPND in our entire sample. This negative finding was also observed in C/C and C/G women but our data suggest that the small number of women homozygote carriers for the G allele display greater [18F]MPPF BPND relative to other individuals. Confirmation of this latter finding, together with the previous studies of 5-HTTLPR polymorphism, indicates that these two polymorphic regions are relevant co-variables of age and gender, which should be considered for future [18F]MPPF studies in normal individuals as well as patients.Overall, our study failed to demonstrate any significant association between C(-1019)G 5-HTThe authors declare that they have no competing interests.18F]MPPF radiosynthesis. AL, NC and SB processed and analyzed the PET data. AL, NC, PG, and PR interpreted the results. AL, PG and PR drafted the manuscript. All authors read and approved the final manuscript.AL coordinated the study. FL participated to the execution of the study. CB carried out the genotype analyses. MA participated to the [18F]MPPF BPND values of 23 women at the peak voxel of the significant cluster in the left superior and middle frontal gyri (a) and in the right and left orbitofrontal cortex (b)Plot of the MPPF BPND values of 23 women at the peak voxel of the significant cluster in the right and left mesial frontal pole (a) and in the right second and third temporal cortex (b)Plot of the [. The first six scans correspond to the women with C/C genotype, the scan number 7 to 21 represent the 15 women with C/G genotype and the two last scans represent the two women with G/G genotype.Click here for file


ACE) genotype as a possible risk factor for migraine (both with and without aura) compared to controls. We also wanted to examine whether a clinical response to an ACE inhibitor, lisinopril, or an angiotensin II receptor blocker, candesartan, in migraine prophylaxis was related to ACE genotype.The main objective of this study was to investigate the angiotensin converting enzyme (ACE insertion (I)/deletion(D) polymorphisms.347 migraine patients aged 18–68 (155 migraine without aura (MoA), 187 migraine with aura (MwA) and 5 missing aura subgroup data) and 403 healthy non-migrainous controls > 40 years of age were included in the study. A polymerase chain reaction (PCR) was performed on the genomic DNA samples to obtain the ACE genotype and allele distributions. Furthermore, there was no significant difference between the controls and the MwA or MoA subgroups.No significant differences between migraine patients and controls were found with regard to ACE genotype or allele frequency and migraine. In addition, ACE genotype in our experience did not predict the clinical response to lisinopril or candesartan used as migraine prophylactics.In our sample there is no association between  Two small open studies reported an improvement of the headache in migraine patients using an angiotensin-converting enzyme (ACE) inhibitor ,2. IndirACE) gene consists of either an insertion (I) allele or a deletion (D) allele forming three possible genotypes: II, ID or DD. Many studies have suggested an association between the ACE-D allele and cardiovascular diseases [ACE polymorphisms and this condition exists  and without (MoA) aura in a Norwegian population.The objectives of the present study were two-fold. Firstly we wanted to examine whether a beneficial effect in the above mentioned migraine prophylactic studies ,5 could lication . SecondlIncluded in the study were 347 migraine patients aged 18–68  and 403The migraine group had a mean age of 41 years (standard deviation (SD): ± 12 years) and consisted of 268 women and 79 men. Median age of migraine onset was 16 years and median attack frequency was 4.0 attacks per month. In the control group with 233 women and 170 men, mean age was 50 years (SD: ± 7 years).311 of the samples were genotyped by AstraZeneca, R&D Genetics, UK, and 439 samples were genotyped at the Department of Neurology, Trondheim University Hospital, Norway.ACE gene. The oligonucleotide primers [DNA was extracted from peripheral EDTA-blood stored at -80°C. The D and I alleles were identified on the basis of PCR amplification of the respective fragments from intron 16 of the  primers ,14 used 2, 1 U AmpliTaq Gold, 200 μM dNTPs and 5 μL DNA template in a total reaction volume of 25 μL, enzyme activation at 94°C for 20 min, denaturation at 94°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 2 min for a total of 32 cycles. Samples analyzed by LightCycler used the FastStart DNA Master SYBR Green 1 mix, which includes Taq DNA polymerase (Roche Diagnostics), plus 3 mM MgCl2, and 2 μL DNA template, in a total reaction volume of 20 μL with enzyme activation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing at 50°C for 5 s, and elongation at 72°C for 15 s, for a total of 35 cycles. The fluorescence intensity of the double-strand specific SYBR Green I is directly proportional to the amount of PCR product formed. Melting curves indicated the respective melting temperatures of the 191 bp and 479 bp fragments to be 84.5°C and 91.8°C respectively, with samples from heterozygotes displaying a peak at both temperatures. Reaction products were confirmed on 2% agarose gel. The ratio between cases and controls was the same for both methods of analysis and blinded control experiments in 10 random patients analysed by the first method were confirmed by the second method.Part of the samples (n = 311) were amplified using a thermal cycler and the products separated on 2% agarose gel. The remaining samples (n = 439) were analyzed using a LightCycler instrument (Roche). Amplification conditions for the first method were 1.2 mM MgClACE I/D polymorphism. The expected genotype proportions were calculated and compared to the observed proportions according to the Hardy-Weinberg law. The significance level was set at p < 0.05. For comparison between groups we used the χ2 test with one or two degrees of freedom. To compare means  we used one-way ANOVA. Statistical analysis were performed using SPSS version 13.0 for Windows . Power calculation for the association between ACE polymorphisms and migraine was performed with the method described by Altman with correction for unequal sample sizes [ACE polymorphisms and drug response, we performed a one sample two tailed test with alpha = 0.05.Observed genotype count was used to calculate genotype and allele frequencies for the le sizes . For theThe study was approved by the regional committee for ethics in medical research, and by the Norwegian data inspectorate. All subjects included gave a written informed consent.ACE-D allele tended to be more frequent (p = 0.058) among responders than non-responders in the candesartan group (Table The observed genotypes in the control population did not deviate significantly from the Hardy-Weinberg equilibrium (p = 0.98). With regard to the genotype and allele distributions, no significant differences between migraine patients and controls were detected, even though the up Table . FurtherFrequencies of the genotypes and alleles for the different studies are presented in Table ACE genotype or allele frequency in a migraine group compared to a control group. Associations between ACE polymorphism and migraine reported in other studies are not consistent and have been detected in different diagnostic or sex categories. The results of these studies are shown in Table ACE allelic frequencies between migraine patients and controls, but stratified by gender the DD frequency was significantly lower in male migraineurs than controls (not included in Table ACE polymorphism and migraine (MoA and MwA) should have >80% power to detect an association of the same magnitude as in the study by Paterna et al [ACE genotype and response to prophylactic drugs influencing the RAS. The allele frequency in the responders versus non-responders in the candesartan group had a p-value of 0.058 and with low numbered groups the risk of a false negative result is present.In the present Norwegian sample, there is no difference in ACE genotype was not to detect a small theoretical association, in which case this subgroup analyses would be underpowered, but to see whether there was an association so strong that it would be valuable in clinical use predicting response in migraine – prophylactic treatment. That is whether it would be clinically beneficial to use ACE genotype to predict whether the patient would respond to the drug or not. Our opinion is that in order for an association to be clinical valuable at least 75–100% of responders to a migraine-prophylactic drug should have a specific ACE-genotype. The power of our study to measure a percentage of 75% DD among the candesartan responders with the control population as reference is > 80%.The purpose of looking at the association between responders in the two clinical trials and A limitation of the study is that the control group was not directly interviewed increasing the risk of migraineurs self-reporting themselves as non-migraineurs and thereby increasing the risk of type II errors. In order to minimize this problem participants in the control group were required to have no other headache condition and less than one headache day per month.ACE-alleles in different populations (Table Population stratification refers to differences in allele frequencies between cases and controls due to systematical differences in ancestry rather than in the association of genes with disease ,18. TherACE genotype distribution between a migraine and a control population in our material. Our study also indicates that ACE genotyping will not be a valuable tool for predicting clinical response of drugs influencing the angiotensin system in headache treatment. It is, however, important that these findings should be confirmed in other studies with more patients and among different ethnic groups.There was no difference in RAS, renin-angiotensin system; ACE, angiotensin converting enzyme; MoA, migraine without aura; MwA, migraine with aura; ARB's, angiotensin II receptor blockers.Co-authors Amanda Gladwin and Katryn Owen are AstraZeneca staff.No disclosures from the rest of the authors.ET, LJS, GB, LRW and HS were involved in designing the study. AG, KO and LRW were responsible for the genotyping. ET and LJS conducted the statistical analyses. All authors were involved in either drafting the manuscript or revising it.The pre-publication history for this paper can be accessed here:


DTNBP1) polymorphisms and schizophrenia. Recently we identified a DTNBP1 SNP (rs9370822) that is strongly associated with schizophrenia. Individuals diagnosed with schizophrenia were nearly three times as likely to carry the CC genotype compared to the AA genotype.A number of studies have found associations between dysbindin  as well as nicotine and opiate dependence but not alcohol dependence. Individuals suffering PTSD were more than three times as likely to carry the CC genotype compared to the AA genotype. Individuals with nicotine or opiate dependence were more than twice as likely to carry the CC genotype compared to the AA genotype.The DTNBP1 in psychiatric conditions and suggests that there is a common underlying molecular defect involving DTNBP1 that contributes to the development of several anxiety and addictive disorders that are generally recognised as separate clinical conditions. These disorders may actually be different expressions of a single metabolic pathway perturbation. As our participant numbers are limited our observations should be viewed with caution until they are independently replicated.This study provides further support for the importance of  DTNBP1 gene in schizophrenia. Linkage studies first identified DTNBP1 as a region of high schizophrenia susceptibility [DTNBP1 have since been identified to be associated with schizophrenia [DTNBP1 is a strong candidate for schizophrenia pathogenesis as it is thought to play a pivotal role in regulating the glutamatergic system [DTNBP1 provide further evidence for its role in schizophrenia aetiology. Reduced DTNBP1 mRNA expression in cerebral cortex has been associated with risk haplotypes for schizophrenia [DTNBP1 mRNA and protein expression has been found in the hippocampal formation and dorsolateral prefrontal cortex of schizophrenia patients [Dystrobrevin binding protein 1 (DTNBP1) is a neuronal protein that binds to alpha- and beta-dystrobrevin in muscle and brain [tibility -4 and poophrenia ,5,6. Bioc system ,8. Glutac system . Functioophrenia . Reducedpatients -13. Schipatients . More repatients . Studiespatients . Tang etpatients  concludeDTNBP1 is associated with other psychotic conditions. Association with DTNBP1 was found with bipolar disorder but only in a small subgroup characterised by the complication of psychotic features during episodes of mood disturbance [DTNBP1 may also be associated with substance-induced psychoses [DTNBP1 variation was identified in a case-control study [turbance . Methampturbance ,17 and asychoses . Signifiol study .DTNBP1 variation shows genetic association with a range of psychiatric conditions, including schizophrenia, suggesting that changes in metabolism related to variations in DTNBP1 protein function underlies a common molecular defect in these conditions. A polymorphism in the human dopamine D2 receptor (DRD2) (rs6277) has previously been found to be associated with a number of psychiatric conditions or traits associated with psychopathology. The DRD2 polymorphism was first found to be associated with schizophrenia [DRD2 polymorphism, a common polymorphism in DTNBP1 may be associated with a range of psychiatric conditions. In a previous study, we found a polymorphism in DTNBP1 (rs9370822) that was strongly associated with schizophrenia [DTNBP1 SNP in addictive and anxiety disorders, a number of psychiatric groups were genotyped for rs9370822.The aim of this study was to examine whether ophrenia . Since tophrenia , workingophrenia , impulsiophrenia  and alcoophrenia . Like thophrenia . It is aophrenia ,26, so, The control group consisted of 250 unrelated Caucasians  with a mean age of 36.8 years (s.d. ± 12.8 years). The control group consisted mostly of medical and nursing staff recruited through hospitals, and university students and academic staff. Formal screening for psychological disorders was not undertaken in the control population. As such the controls represent an unselected control group.A total of 120 unrelated Caucasian participants  diagnosed as opiate dependent were recruited for this study, all of whom were being assessed for naltrexone treatment as outpatients of the Hospital Alcohol and Drug Services Unit of the Royal Brisbane and Women's Hospital. Participants had a mean age of 28.7 years of age. Approximately half of the participants were being managed on methadone prior to detoxification (47.2%) while the other half were on heroin (52.8%). Those on methadone had a mean dose of 48.1 milligrams (s.d. 30.5), with a range between 10-165 milligrams. The mean age of onset of heroin use was 22.4 years of age (s.d. 5.14), with a range between 15-43 years. Mean number of participant-reported detoxifications prior to this occasion was 3.5 (s.d. 3.3), with a range between 0-16 detoxifications. Cannabis was the most commonly concurrently used illicit substance reported by participants prior to treatment (52.5% reported use), followed by amphetamines (14.9% reported use).A total of 127 unrelated male Caucasian patients diagnosed with PTSD using Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria were recruited through the Greenslopes Private Hospital for the study. All subjects were Vietnam combat veterans who had served in the Australian Armed Forces with a mean age of 52.3 years (s.d. ± 6.1 years). None were being treated with psychotropic medications. Patients were excluded from the study if they had a diagnosis of psychosis, bipolar disorder, obsessive-compulsive disorder, or organic brain syndrome including dementia. All subjects had sufficient comprehension of English and could understand the administered questionnaires. Patients were assessed for PTSD by a consultant psychiatrist or a senior psychiatric registrar using DSM-IV criteria. Furthermore, every patient exceeded the clinical cut-off score of 94 on the Mississippi Scale for combat-related PTSD [n = 65) of these patients being diagnosed with two or more alcohol related medical conditions such as pancreatitis, cirrhosis, hepatitis or peripheral neuropathy. Alcohol dependent patients were excluded from the study if they had dementia, delirium, psychosis, or any other condition that would affect their ability to provide informed consent.A total of 231 unrelated Caucasian  alcohol dependent subjects were recruited from large public hospitals in Brisbane, Australia. The mean age of the group was 42.1 years (s.d. ± 10.8 years). All subjects were assessed by a clinical psychologist experienced in drug and alcohol dependence and met DSM-IV criteria of alcohol dependence disorder. All were inpatients and represented a spectrum of severity with a significant proportion  unrelated Caucasians with a mean age of 43.3 (s.d. ± 11.1 years) were recruited for this study through hospital and media advertisements. Participants were 18 years of age or older and had smoked for at least three years and were generally healthy despite currently smoking 15 cigarettes or more per day. All were motivated to reduce smoking and had the goal of eventual cessation. However, all participants had at least one serious but unsuccessful attempt at quitting in the previous 24 months. One hundred and thirty nine of the participants were administered the Fagerstrom test for nicotine dependence .To minimise population stratification bias, both control and clinical subjects were recruited in the Brisbane region  and all were of British or European descent.Ethics approval was obtained from all institutions involved and each participant gave written informed consent. This study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).Oragene kits were used to extract DNA from saliva samples. Samples were genotyped using a homogeneous MassEXTEND (hME) Sequenom assay performed by the Australian Genome Research Facility. The hME assay is based on the annealing of an oligonucleotide primer (hME primer) adjacent to the SNP of interest. The addition of a DNA polymerase along with a mixture of terminator nucleotides allows extension of the hME primer through the polymorphic site and generates allele-specific extension products, each having a unique molecular mass. The resultant masses of the extension products are then analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and a genotype is assigned in real time. The hME assay was performed in multiplex with up to 36 reactions in a single well. Genotyping failure rates varied between two and five percent in the different groups.2 test was performed to identify statistical associations between alleles and genotype and schizophrenia status. Odds ratios (OR) were also calculated. Tests were performed on both genotype and allele data. Statistical tests were performed using the COMPARE2 program from the WinPepi suite of epidemiology programs [A Pearson's χprograms  and SPSSprograms . The anaDTNBP1 SNP, rs9370822, in a range of psychiatric conditions, it was genotyped in 250 controls, 127 PTSD subjects, 147 nicotine dependent subjects, 120 opiate dependent subjects and 231 alcohol dependent subjects. In addition to our previously reported association with schizophrenia, rs9370822 was found to be associated with PTSD, opiate dependence and nicotine dependence at the allele level but not with alcohol dependence .Examination of the genotype odds ratios suggests that, like PTSD, a partially dominant mode of inheritance is operating for nicotine dependence. However, when compared to the low-risk homozygote, the odds ratio of the other two genotypes indicated that either a very weak partially dominant mode of inheritance or, more likely, a C-allele-recessive pattern of inheritance for schizophrenia and opiate dependence was present, i.e. both the heterozygote OR and the OR for the low-risk homozygote were approximately one. The rs9370822 SNP was found to be associated with schizophrenia and opiate dependence when analysed under a recessive model by pooling genotypes .Alcohol dependence was not found to be associated with rs9370822 at the genotype level or under any inheritance model. All sample groups were determined to be in Hardy-Weinberg equilibrium based on the respective genotype frequencies of each group .In order to identify a common molecular mechanism of disease etiology, us study . To replOur schizophrenia study was the first to identify rs9370822 to be associated with a psychiatric condition . The posThe relationship between abnormal dysbindin function and addictive and anxiety disorders is unclear. Our findings may indicate that the rs9370822 polymorphism affects susceptibility to addictive and anxiety disorders by implication of glutamatergic neurotransmission or the dopamine system. We previously identified rs9370822 to follow a recessive pattern of inheritance with respect to schizophrenia risk  which isDTNBP1 may affect the dopamine pathway as dysbindin overexpression decreases dopamine release and suppression of dysbindin expression increases dopamine release in the mouse in vitro [It has also been reported that variation in in vitro . A studyin vitro .DTNBP1 polymorphism identified in this study is possibly involved in psychiatric liability not only for schizophrenia but also for addictive and anxiety disorders. It is possible that DTNBP1 variation affects a common pathway involved in anxiety and addictive disorders. Whether this pathway involves dopamine or glutamate signalling or both, remains to be elucidated. As our participant numbers are limited our observations should be viewed with caution until they are independently replicated. However, the associations are unlikely to occur by chance due to sample size as five separate groups were analysed (including schizophrenia in a previous study) and four have been found to be significantly associated with rs9370822. Although this SNP is intronic, it has been found to be strongly associated with schizophrenia, PTSD, opiate and nicotine dependence. Future studies may explore whether this SNP is functional and what effect it has on DTNBP1 functioning in the brain.The Our findings are at odds with current knowledge as these conditions are generally regarded as separate clinical entities. However, our data suggests that these conditions may be different phenotypic expressions of the same fundamental molecular defect in either the dopamine or glutamate pathways, or both. Supporting this idea, a recent review of genome-wide association studies in schizophrenia  suggesteThe authors declare that they have no competing interests.JV: Involved in conception and design, acquisition of data, analysed and interpreted data, drafted the article and approved final version. CS: Involved in conception and design, acquisition of data, critically revised article and approved final version. IH: Involved in conception and design, acquisition of data, analysed and interpreted data, critically revised article and approved final version. JC: Involved in acquisition of data, critically revised article and approved final version. BL: Involved in conception and design, acquisition of data, critically revised article and approved final version. RY: Involved in conception and design, acquisition of data, critically revised article and approved final version. CM: Involved in conception and design, acquisition of data, critically revised article and approved final version.


Some studies have raised the question about whether the good results obtained with the Charnley prosthesis could be replicated at general hospitals when it comes to the frequency of early complications and failure rates, both of which would be higher than those published by centres devoted to hip arthroplasties.We reviewed the results of 404 Low Friction Arthroplasties of the hip implanted between 1976 and 1993 in a general hospital by general orthopaedic surgeons. For the survival analysis, the end-point chosen would be the chirurgical revision of any of the prosthetic components for whatever reason.The complications were 16 dislocations (4%), 14 deep infections (3.5%), 2 neurological injuries  and 5 clinical deep venous thromboses (1.2%) (2 pulmonary embolisms). The survival rate at 25 years, both for stem and cup, was 83%. Survival was higher in those arthroplasties implanted in patients older than 60 years, with statistical significance.Low Friction Arthroplasty undertaken at general hospitals by general orthopaedic surgeons feature similar outcomes to those found in centres devoted to hip surgery. Low Friction Arthroplasty (LFA) is still considered the "golden guide" when it comes to compare the different Total Hip Arthroplasy (THA) models. Many studies have been published on LFA, showing excellent, long-term results, both about clinical and radiological issues ,2 as welOur Unit belongs to a general hospital located in northern Spain, and it serves a population of 180,000; it is not specifically devoted to hip surgery. Since its opening in 1976, the hospital has remained faithful to LFA throughout the years  performed as primary procedures consecutively from 1976 to 1993 have been included in this study. In all cases the implant used was the prosthesis designed by J. Charnley, with the technical and design improvements added throughout the years [Throughout 2004, two independent observers who did not participate in the surgery (ASV and JFL) reviewed the medical histories and radiographies of each patient, categorizing the radiographs into those that were possibly, probably or definitely loose. General data about the patient and the intervention as well as about the clinical development were gathered. Since we are dealing with a long-term, retrospective study, only easily verifiable variables  were accepted. Any complications which required an active medical intervention and which, accordingly, were featured in the medical histories, were also included.For the survival analysis, the end-point chosen would be the chirurgical revision of any of the prosthetic components for whatever reason. Variable data  were recorded in a SIGMASTAT v 3.00 for Windows  database built for such purpose. The survival calculations were done with this tool, following the Kaplan-Meier method  with a cThe mean age at the moment of surgery was of 67 years (SD: 8.8), where the maximum age was 91 years and the minimum was 36. 57% of the cases were males and 43% females; 45% were right side and 55%, left side.The following complications were registered: 16 dislocations (4%), 14 deep infections (3.5%), 5 periprosthetic fractures (1.2%), 2 neurological injuries , 5 deep venous thromboses (1.2%) with 2 pulmonary embolisms, one of them fatal.After 25 years, the cup and stem survival rates were of 83% Table  and 2, (A surgical technique is reproducible if its results are similar in real, non-ideal, conditions, and that is what seems to happen with LFA. In the long term, the survival rate of LFA in our centre is very similar to those shown in historical series or arthroplasty register ,12 and iBoth in our series as well as in a multicentric series , the gloThe dislocation rate is also higher than the classical rate of 2%–3% for primary surgery  being clThe assessment of results in THA requires a multiple approach, either through clinical score sets complemented with imaging analyses, curves or survival analysis, or through score and review indices about the quality of life related to health. Each approach addresses a requirement of the complex evaluation of this technique which, paradoxically enough, must be reviewed in the long term in spite of being relatively new and rapidly evolving in developed countries.There remain many unresolved questions in hip replacement. Well-constructed clinical tests offer the best hopes of answering them. Such tests are onerous, and time and money are required to collect, record and analyze the data. The relatively few clinicians who undertake such tests must have an understanding of the statistical methods used and the problems they may encounter. It is especially difficult to find series where you can compare long term results in the literature and whether the arthroplasty was implanted in general hospitals. Only retrospective studies are available, which must be accepted in spite of the known issues with validity. The methodological flaws common to these studies could be alleviated using, as in our case, data about which there is absolute certainty they are true, even though this would mean an obvious loss of information. In joint replacement surgery the usual end-point is the decision to remove the prosthesis and revise or convert it to some other form of treatment. This end-point has been criticized because the criteria used to decide the need for removal will differ between patients and surgeons. This form of analysis defines a failure point or terminal event and provides an assessment of not only how many failures there had been but also how long after the operation did the failure occur. It does not measure function or pain, unless they are included in the definition of failure Figure . Using aTo sum up, our study seems to confirm a higher frequency of early complications and a higher assessment rate of the THA in general hospitals compared to those in dedicated centres, although in the long term this trend does not keep up and the survival rates become similar. LFA undertaken at general hospitals by general orthopaedic surgeons feature similar outcomes to those found in centres devoted to hip surgery. A national joint replacement registry would be a useful tool to compare outcomes for high and low volume community hospitals as well as academic centres.Low Friction Arthroplasty undertaken at general hospitals by general orthopaedic surgeons feature similar outcomes to those found in centres devoted to hip surgery.THA: Total hip arthroplasty; LFA: Low friction arthroplasty.The authors declare that they have no competing interests.DHV conceived of the study, participated in its design and coordination and drafted the manuscript, ASV and JFL reviewed the medical histories and radiographies. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Survival tables. 3 tables.Click here for file


The area of atherosclerotic lesions in the coronary arteries was quantitated, and coronary narrowing was measured. The occurrence of myocardial infarction (MI) was assessed. Genotyping was by restriction endonuclease analysis. Men carrying the minor C allele had larger areas of complicated lesions (P = .024) and a higher number of coronary arteries that had over 50% stenosis (P = .036) compared to men representing the common GG genotype. The COX-2 polymorphism was not associated with MI. Our data suggest that COX-2 may be involved in plaque growth.Cyclooxygenase (COX) catalyzes formation of prostaglandins that contribute to the inflammation in atherosclerosis. Our objective was to study whether the functional C variant of the −765G→C polymorphism in the human COX-2 gene associates with the severity of coronary atherosclerosis measured at the coronary artery level. The Helsinki sudden death study autopsy material ( Cyclooxygenase (COX) catalyzes thefirst two steps in prostanoid production from arachidonic acid to manyprostaglandins including prostacyclin and thromboxane. The enzyme has threeknown isoforms. COX-1 is constitutively expressed in most human tissues underbasal conditions. COX-2 expression is primarily induced in response toinflammatory stimuli by growth factors, mitogens, and cytokines [2), which acts as a vasodilator and inhibits plateletaggregation, leukocyte activation, and adhesion [COX-2 levels areraised in chronic inflammatory diseases including atherosclerosis. COX-2expression has been detected in endothelial cells, smooth muscle cells,monocytes and macrophages within human atherosclerotic lesions , 4. Manyadhesion .A functional G→C polymorphismlocated 765 basepairs upstream from the transcription start site (−765G→C) has been identifiedin the human COX-2 gene with C allele leading to decreased promoter activity invitro . PreviouThe previous contradictory observations suggest that additional evaluation is warranted to investigate the associationbetween the COX-2 765G→C polymorphism and cardiovascular disease. We have examined the association of thispolymorphism with the risk of severity of atherosclerosis at the coronaryartery level in a previously collected autopsy series of a genetically homogeneouspopulation of Finnish men who had died suddenly out of hospital.n = 115), other diseases in 20.3% (n = 61) and violent death (suicides and accidents) in 41.4% (n = 124).Men with a coronary event  (n = 72) were also comparedwith men with no coronary event (n = 228).HSDS was designed to investigate factorspredisposing to sudden death in Finnish middle-aged men living in Helsinki and itsenvironment . The autAt autopsy, coronary angiography was performedusing vulcanizing liquid silicone rubber mixed with lead oxide as contrastmedium . ProximaThe proximal parts of the LAD, LCX,and RCA were dissected free, opened, and attached to a card and then fixed in10% buffered formalin. The vessel wall was subsequently stained for fat by theSudan IV method. The following atherosclerotic changes were assessed: fattystreak, fibrous plaque, complicated lesion, and the area of calcification. Flator raised intimal lesions that were distinctly stained by Sudan IV and did notshow more complex changes were classified as fatty streaks. An elevated lesionthat did not display ulceration, haemorrhage, necrosis, or thrombosis wasconsidered as a fibrous plaque. An area was regarded as complicated lesion, ifit expressed one or several changes mentioned above, with or without calciumdeposit. The part of the aorta showing intense X-ray-positive signal in theradiogram was considered as an area of calcification. The areas ofatherosclerotic lesions and the total areas of coronary segments were evaluatedusing the standard planimetric technique . The proAutopsy and COX-2 genotype data were available in 300 cases, thesecomprising the final study population.A spouse, relative, or a close friend of thedeceased was interviewed within 2 weeks following the autopsy. Besides the questionspertaining to risk of sudden death ,additional questions were included to define the smoking habits of thedeceased. The relative/friend was asked whether the person had smoked duringhis life and how many cigarettes he had smoked daily. Data on smoking habitswere obtained in 148 cases. In addition to COX-2 genotype and autopsy data, complete data on allrisk factors were available in 118 cases.http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).The 25 μl-reaction was composed of 50 pmol of each primer  , 200 μM of each dNTP and 2,5 U HotStarTaqDNA Polymerase in 1 × PCR buffer . PCR conditionsincluded 15 minutes of initial polymerase activation step at 94°C followed by35 three-step cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 5 minutes.The promoter region of the human COX-2gene surrounding the site of −765G→C polymorphismwas polymerase chain reaction (PCR) amplified using DNAs extracted from cardiacmuscle as a template. Primers for DNA amplification were designed based on thepublished sequence of the human COX-2 gene  using the Primer3 software restriction endonuclease. PCR product was cleaved into fragments of 168 bp and60 bp, if the G allele was present. Digested products were resolved with 2%MetaPhor  agarosegel electrophoresis and visualized by ethidium bromide staining.Results were analyzed by SPSS forWindows software, version 14.0 . Mean ± standard error (SE) is reported for continuous variables. For normallydistributed continuous variables, one-way-ANCOVA was used with age, body massindex (BMI), smoking status (yes/no), hypertension (yes/no), and diabetes(yes/no) as covariates. Since variables measuring atherosclerotic changes werenot normally distributed, logarithmic transformations were utilized for valuesof fatty streaks, fibrous plaques, calcifications, and complicated lesions, butresults are displayed in crude form.In the study population, COX-2 genotype frequencies were GG 74.4%, GC 24.3%, and CC1.3%. Since there were only four cases representing the CC genotype, mencarrying the C allele were pooled together as one group for statisticalanalyses.P = .024) with adjustment for ageand BMI. After further adjustment with smoking, hypertension, and diabetes,there was a significant difference between genotypes and calcifications (P = .031) and genotypes and complicated lesions (P = .017) (P = .036) comparedto those with the GG genotype, with adjustment for age and BMI. After furtheradjustment with smoking, hypertension, and diabetes, this difference was evenmore significant (P = .008)  . No sign = .008) . There w = .008) .n = 72) were also compared with men with nocoronary event (n = 228). In the coronary event group, mencarrying the C allele had significantly larger areas of complicated lesions intheir coronary arteries than the men with GG genotype with adjustment for ageand BMI (P = .040) and after further adjustment with smoking,hypertension, and diabetes (P = .015). Also in the coronary event group,the C allele carriers had a higher number of over 50% stenosis in one, two, orthree major coronary arteries comparedto those with the GG genotype, with adjustment for age and BMI (P = .040) and after furtheradjustment with smoking, hypertension, and diabetes (P = .050)  ( = .050) .No sign = .050) .The results of this study propose that middle-aged Finnishmen with sudden death that carried the rare Callele of the COX-2 765G→C polymorphism hadmore often advanced coronary plaques, characterized by more extensive areas ofcomplicated lesions, compared to men with the GG genotype. This association wasmost pronounced in subjects with a coronary event. Variation in COX-2 promoter genotypeshowed no association with early coronary atherosclerosis characterized by fattystreaks and fibrous plaques. A previous study has shown that the 765G→C allele mightprovide protective effects against myocardial infarction . C allelMen carrying the C allele had a higher number of coronary arteries that had over 50% stenosis,compared to men who were GG homozygous. In contrast, the relation between the Callele and severity of atherosclerosis has previously been studied at thecoronary level by angiography in an Italian population, but no association wasfound . The pol2 whichis generated through COX-2 route. Asa consequence of diminished PGE2 biosynthesis, a decrease in MMP-9expression could follow [The finding that stenosiswas more often observed in men possessing C allele than in men representing GGgenotype may provide support for the hypothesis that C allele may lead to moreextensive plaque growth. In fact, since the C allele leads to decreasedpromoter activity , it could follow  and leadd follow reportedd follow , 19.In conclusion, the rare COX-2 gene C allele associated with complicatedplaques and severe stenosis at the coronary artery level. This suggests thatCOX-2 may be involved in plaque growth. No protective effect against MI wasseen.


Mitochondrial function is impaired in Parkinson's disease (PD) and may contribute to the pathogenesis of PD, but the causes of mitochondrial impairment in PD are unknown. Mitochondrial dysfunction is recapitulated in cell lines expressing mitochondrial DNA (mtDNA) from PD patients, implicating mtDNA variants or mutations, though the role of mtDNA variants or mutations in PD risk remains unclear. We investigated the potential contribution of mtDNA variants or mutations to the risk of PD.We examined the possibility of a maternal inheritance bias as well as the association between mitochondrial haplogroups and maternal inheritance and disease risk in a case-control study of 168 multiplex PD families in which the proband and one parent were diagnosed with PD. 2-tailed Fisher Exact Tests and McNemar's tests were used to compare allele frequencies, and a t-test to compare ages of onset.The frequency of affected mothers of the proband with PD  was not significantly different from the frequency of affected females of the proband generation  . After correcting for multiple tests, there were no significant differences in the frequencies of mitochondrial haplogroups or of the 10398G complex I gene polymorphism in PD patients compared to controls, and no significant associations with age of onset of PD. Mitochondrial haplogroup and 10398G polymorphism frequencies were similar in probands having an affected father as compared to probands having an affected mother.These data fail to demonstrate a bias towards maternal inheritance in familial PD. Consistent with this, we find no association of common haplogroup-defining mtDNA variants or for the 10398G variant with the risk of PD. However, these data do not exclude a role for mtDNA variants in other populations, and it remains possible that other inherited mitochondrial DNA variants, or somatic mDNA mutations, contribute to the risk of familial PD. ND5 gene mutations have been associated with PD [Mitochondrial complex I is impaired in platelets  and in t with PD ,23, but  with PD -31. A co with PD ,32; howe with PD ,24. ThusWe sought to investigate the association of mtDNA variants with the risk of PD and with the age of onset of PD by taking advantage of a unique cohort of familial PD subjects. We focused on the subset of families in which the proband and either the mother or the father were diagnosed with PD. This allowed us to test for a maternal bias in the inheritance of PD and also to test whether any of the common mtDNA haplotypes are risk factors for disease.As part of an ongoing genetic study recruiting siblings diagnosed with PD, 654 multiplex PD families were ascertained. All available affected individuals were seen by a movement disorder specialist at one of 59 Parkinson Study Group sites. A standardized clinical assessment was completed including the validated Unified Parkinson's Disease Rating Scale (UPDRS) Parts II & III . A diagnparkin mutations (homozygous or compound heterozygous) or a LRRK2 mutation. Families in which both the mother and the father were diagnosed with PD were also excluded from analyses (n = 11).To explore the role of inherited mitochondrial factors, a subset of families was analyzed which included a proband with PD that was verified by direct clinical examination and with either a mother or a father (but not both) diagnosed with PD (n = 168). These 168 families excluded families with either two Control samples (n = 895) were obtained from the NINDS Human Genetics Resource Center at the Coriell Institute Coriell Cell Repositories . These control samples as well as a single individual from each of 488 of the Parkinson's Research: The Organized Genetics Initiative (PROGENI) families were genotyped at the Center for Inherited Disease Research (CIDR) using the Illumina HumanCNV370 version1_C BeadChips  and the Illumina Infinium II assay protocol as part Cases and controls were matched in four dimensional space  using the following algorithm. Gender was coded as 0 and 1, the age distribution was normalized and multiplied by 4, and each of the first two MDS components were normalized and multiplied by 8. The Euclidean distance between all possible cases and all possible controls was then computed, and the minimum distance to a control was determined for each case. The case-control pair with the minimum distance was flagged and declared a final match. The selection process was then repeated iteratively with the remaining individuals, flagging the pair with the minimum distance, until all cases were matched to a control. The distribution of distances between cases and their final matched control sample was reviewed and those pairs that were beyond 3 standard deviations from the mean were not included in the paired analyses (n = 10). The weights used  were selected to balance the contribution of each factor and minimize the number of outliers; without these weights, gender dominated the matching process at the expense of the ethnicity components. Demographics of cases and controls are indicated in Table To allow haplogroup identification, 12 SNPs were genotyped. These included 10 SNPs genotyped in previous reports that are associated with particular haplogroups : 1719A  is determined by the sign test  instead of McNemar's test.Age at onset was compared between carriers and non carriers of each variant using an independent t-test. Since all siblings share the same mitochondrial SNP alleles, and therefore haplogroups, we repeated these analyses substituting the mean age at onset of all affected siblings in a nuclear family for the age at onset of the individual genotyped. The number of affected siblings in a family ranged from 1 to 5.A total of 168 families were identified in which one parent and one or more offspring were affected with PD, with at least one offspring having had their PD diagnosis verified by direct clinical examination. There were 259 siblings among the offspring generation of these families, of which 115 (44.4%) were females. Among the 168 parents with PD, 83 (49.4%) were females (mothers), which was not significantly different compared to the offspring generation (2-tailed Fisher exact p = 0.32).Next, we assessed the association of mitochondrial haplogroups with the risk of maternal transmission of PD. The most common European mitochondrial haplogroups were defined as shown in Table When allele frequencies were compared between cases and controls Table , haplogrThis study of the PROGENI subjects represents the first to report data on mitochondrial genetic variants in conjunction with maternal inheritance in a large group of familial PD cases, and fails to reveal a maternal bias in the inheritance of familial PD. The question of maternal inheritance in PD has been controversial, with some studies reporting a bias towards maternal inheritance in PD ,6, and oPARK2 or LRRK2 genes (either two PARK2 mutations or one LRRK2 mutation), [PINK1 and DJ1, but such mutations are relatively rare [The current study failed to detect a significant bias towards maternal inheritance in familial PD, adding additional uncertainty regarding the role of inherited mtDNA variants in PD. A potential concern for this study is that the stronger genetic influence in familial PD cases might have led to a closer to 50:50 ratio of males to females in contrast to the male predominance for sporadic PD. However, this observation is unlikely to be a factor in this study as siblings and parents came from the same families. To improve the ability to detect an influence of mtDNA genetic variants, all families with known causative mutations in the tation), ,43 were ely rare .in utero environmental factors[Mitochondrial genes are passed on from mothers to offspring but not from fathers to offspring. Therefore, to the degree that heritable mitochondrial genetic factors influence the risk of PD, there should be a greater risk of PD among offspring of women with PD compared to offspring of men with PD. However, alternative mechanisms also could result in a maternal inheritance bias. One example is epigenetic changes (imprinting) of autosomal genes. An additional potential mechanism is l factors, as suggl factors,47. AnotIndirect data has implicated mtDNA in the mitochondrial complex I defect in PD. "Cybrid" cell lines expressing mtDNA from PD patients manifest impaired mitochondrial complex I activity ,10, suggAnother way to test a role for mtDNA variants in PD, in addition to testing for a maternal inheritance pattern, is to compare the frequencies of mtDNA variants between cases and controls. Prior studies of mitochondrial variants in PD have failed to identify clearly pathogenic mutations in the vast majority of patients ,20, but Reports of a possible association of haplogroups J and K with PD have been inconsistent ,27,29-32We did not detect a maternal bias in the inheritance of PD among PROGENI subjects, and failed to confirm previously reported associations of inherited mtDNA variants with PD risk. This suggests that other factors, possibly including somatic mtDNA mutations, may account for the complex I defect in PD.The authors declare that they have no competing interests.DKS conceived of the study, participated in its design, data analyses and interpretation, and drafted the manuscript; NP participated in the design of the study, performed the statistical analyses, and aided in interpreting the results and helped to draft the manuscript; DKK and MWP assisted in the design of the molecular studies and performed the laboratory experiments; CAH coordinated the recruitment, initial screening and follow-up of the subjects; AR participated in the design of the overall recruitment portion of the study and ensured regulatory oversight at the sites. RP participated as Chair of the Steering Committee; WCN assisted in planning the study and conducted the genotyping; TF conceived of the study, participated in its design and coordination and in analysis and interpretation of the results, and helped to draft the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/53/prepub