Asthma is a complex genetic disease with more than 20 genome-wide scans conducted so far. Regions on almost every chromosome have been linked to asthma and several genes have been associated. However, most of these associations are weak and are still awaiting replication.In this study, we conducted a second-stage genome-wide scan with 408 microsatellite markers on 201 asthma-affected sib pair families and defined clinical subgroups to identify phenotype-genotype relations.The lowest P value for asthma in the total sample was 0.003 on chromosome 11, while several of the clinical subsets reached lower significance levels than in the overall sample. Suggestive evidence for linkage (p = 0.0007) was found for total IgE on chromosomes 1, 7 and again on chromosome 11, as well as for HDM asthma on chromosome 12. Weaker linkage signals could be found on chromosomes 4 and 5 for early onset and HDM, and, newly described, on chromosome 2 for severe asthma and on chromosome 9 for hay fever.This phenotypic dissection underlines the importance of detailed clinical characterisations and the extreme genetic heterogeneity of asthma. Many chromosomal regions have been shown to be linked or associated to asthma and asthma-associated traits in humans . More reThe mainstay of all genetic studies has been genome-wide linkage scans in families with at least two asthma-affected siblings. Based on a previous analysis of a genome-wide scan of asthma ,8 with i97 families consisting of at least two children with confirmed clinical asthma were collected during the first stage of the German genome scan ,Frequent replication, however, may point toward lead genes. Most regions listed in table The locus on chromosome 12 with suggestive linkage for HDM in our study is known from many other reports. Also our first scan showed weak evidence for linkage in this region at D12S351 (p = 0.01) , whereasOn chromosome 2, we found a locus in families with severe asthma that has not yet been described in other studies. A p-value of 0.007 has already been found in our first genome-wide scan [The German "summer-type" locus on chromosome 9 is also not frequent in other studies. This might be due to the fact that many, but not all, genes are necessary for a common trait. Our first asthma scan has already shown linkage in this region with a p-value of 0.007 for D9S1784 [We conclude that this phenotypic dissection is a useful tool to detect linkage in a heterogeneous disease like asthma because some of the sub phenotypes reached even better significance values than the main trait. We show that the precision of the phenotype can be more effective than expanding the sample size only. Unfortunately, large sample sizes are needed to assure at least moderate sample size in subsets. Grouping by early onset or disease severity could be applied to almost every complex disease, but for a more specific dissection, clinical expert knowledge is required. A prior analysis of clinical data can help to identify symptom clustering, which -if consistent within families- can reduce genetic heterogeneity.The author(s) delcare that they have no competing interests.MW, JA, and CS were involved in the study design, JA organised the sample collection, conducted the genetic analysis, and prepared the manuscript, MW organized funding and supervised the study. YAL and PN organised and helped with genotyping and revised the article critically for important intellectual content. SL, FR and MW carried out the statistical analysis and made substantial contributions to its conception and design, CS, DB, FF, HJ, JK, AK, AS, MS, WW, PW assisted in the recruitment of families and examined patients and therefore made substantial contributions to the acquisition of data, asthma severity grades are part of the thesis of CS. GS was responsible for the IgE analysis.The pre-publication history for this paper can be accessed here:


The relationship between elevated blood pressure and cardiovascular and cerebrovascular disease risk is well accepted. Both systolic and diastolic hypertension are associated with this risk increase, but systolic blood pressure appears to be a more important determinant of cardiovascular risk than diastolic blood pressure. Subjects for this study are derived from the Framingham Heart Study data set. Each subject had five records of clinical data of which systolic blood pressure, age, height, gender, weight, and hypertension treatment were selected to characterize the phenotype in this analysis.p = 0.00002) evidence for linkage was found between this normalized phenotype and a region on chromosome 1. Similar linkage results were obtained when we estimated the phenotype while excluding values obtained during hypertension treatment. The use of linear mixed models to define phenotypes is a methodology that allows for the adjustment of the main factor by covariates. Future work should be done in the area of combining this phenotype estimation directly with the linkage analysis so that the error in estimating the phenotype can be properly incorporated into the genetic analysis, which, at present, assumes that the phenotype is measured (or estimated) without error.We modeled systolic blood pressure as a function of age using a mixed modeling methodology that enabled us to characterize the phenotype for each individual as the individual's deviation from the population average rate of change in systolic blood pressure for each year of age while controlling for gender, body mass index, and hypertension treatment. Significant ( The relationship between elevated blood pressure and cardiovascular and cerebrovascular disease risk is well accepted. The lifetime risk estimate for developing hypertension is 90% in patients who are 55 and 65 years old . Both syIn examining the effect of systolic blood pressure on cardiovascular disease, it is beneficial to utilize subjects' measurements taken over a period of time. This is because multiple longitudinal blood pressure measurements or long-term average blood pressure are more representative of a person's average or true blood pressure than a single measure for risk assessment . Mixed mEvidence shows that one's genetic makeup is a strong determinant of their risk of developing hypertension. Families prone to higher systolic and diastolic blood pressures also show higher rates of blood pressure increase with age; however, few other studies have explored rate of blood pressure increase . The pur. Of the 1671 subjects in Cohort 2, 1590 were included in the linear mixed model analysis, of which the 1308 with genotype data were included in the linkage analysis. Subjects were excluded from the linear mixed model analysis if only one systolic blood pressure measurement was done.Subjects for this study are derived from Cohort 2 of the Framingham Heart Study Data Set. Cohort 2 was selected because it comprises offspring, thereby providing a higher proportion of siblings than Cohort 1, which is the original cohort with complex family structures. For details on Cohort 2 subject selection, exclusion, distribution, and methodology please refer to In the Cohort 2 data set, each subject has five records of clinical data including but not limited to age, height, weight, systolic blood pressure, cholesterol, fasting glucose, and behavioral risk factors. The variables selected for analysis in linear mixed models were systolic blood pressure, age, height, weight, gender, and hypertension treatment. The selection of these variables was based on past studies investigating similar hypotheses ,7 and beAlso considered was the impact of hypertension treatment on the systolic blood pressure readings. Preliminary graphs of individual systolic blood pressure data indicate that subjects on medication have less linear data than subjects not on medication. While subjects who received treatment make up most of the individuals with high blood pressure, we were concerned that measurements from individuals on medication may be biased. Therefore, we defined the phenotype in two ways: 1) we adjusted the phenotype for hypertension treatment (as described above) and 2) we set all blood pressure measures taken while the individual was receiving treatment to missing. We then conducted analyses on both phenotypes using the same methods and presented the results separately for comparison.The linkage analysis phenotype for each individual is the best linear unbiased predictor of the individual's deviation from the population rate of change in SBP for each year of age while controlling for hypertension treatment, sex, and BMI. The SBP for each individual was measured every 4 years as part of the Framingham Heart Study. The measurement data are not complete in every individual and missing values exist. Given the longitudinal nature of the data and the existence of missing data, linear mixed models were utilized for the slope generation. Linear mixed models also allow for selected covariates to be adjusted for in the analysis such as whether the subject was taking hypertension medication. The model form is described below.Yi = Xiβ + WiΓ + Zibi + εI  ,wherei: Ythe vector of systolic blood pressures for individual iβ: vector containing the intercept and slope of the regression of systolic blood pressure on agei: Xdesign matrix associated with βΓ: vector containing regression coefficients on covariates gender, BMI, and hypertension treatmenti: Wdesign matrix associated with covariates gender, BMI, and hypertension treatmenti: bvector of random effects due to age and BMIi: Zdesign matrix associated with random effects due to age and BMII: εrandom error vector for each individual.Assumptions for the random elements are:i ~ i.i.d. N.The εi ~ i.i.d. N.The bi are independent.The ε and b1, was the phenotype in our genetic analysis. The phenotype for each individual was interpreted as the individual's deviation from the population average rate of change in SBP for each year of age while controlling for gender, BMI, and hypertension treatment. Lastly, the phenotype was converted into a Z-score value for use in the S.A.G.E. 4.2 program [Using SAS, we modeled SBP as a function of age using mixed modeling methodology that enabled us to estimate the population average curve as well as the subject-specific curves based on best linear unbiased predictors. The regression model for each individual was adjusted for gender, BMI, and hypertension treatment. The best linear unbiased estimator of the random effect of the regression coefficient on age, b program .A secondary analysis excluding hypertension treatment was conducted to compare with results acquired when adjusting for treatment. To do this analysis, if a SBP measurement was taken while the subject was taking hypertension medication, the SBP measurement was converted to a missing value. Mixed models were then used with the new SBP measurements to get the new phenotype of individual's deviation from the population average rate of change in SBP for each year of age while controlling for gender and BMI. This phenotype was also converted to a Z-score value for use in the S.A.G.E. 4.2 program .Linkage analysis using sibling pairs can be defined by the regression of the squared sib-pair trait difference on the estimated proportion of alleles siblings share identical by descent . The SIBInitially, the pedigree data set was run through the Identical by Descent4.2 (IBD) procedure of SAGE using the single-point option. The resulting IBD file along with a pedigree file and a parameter file were utilized in the nonparametric linkage analysis of SIBPAL4.2 to identify genetic linkage on the basis of sib-pair phenotypes as defined by the mixed model results. Only full sibs were used in this sibship analysis.Descriptive statistics of normalized phenotype values are shown in Table Significance of the linkage results was evaluated using the criteria of Lander and Kruglyak . With adWhen SBP values taken during hypertension treatment are excluded, the same region on chromosome 1 shows significant or suggestive evidence for linkage to SBP rate of change, as indicated by the bold text in Table In this study, a linear mixed modeling methodology was used to identify SBP phenotypes for each subject. The use of mixed models was appropriate here given the longitudinal nature of the data. By using linear mixed models with best linear unbiased predictors, the phenotype of rate of change can be defined as the slope for each subject while adjusting the values for other covariates. Studies have examined various methodologies for using longitudinal data to identify individual phenotypes ,10 and tFor example, Levy et al.  defined We found significant evidence for linkage of SBP rate of change to a region on chromosome 1 ~188–269 cM). Interestingly, we found similar linkage results regardless of whether we excluded values under hypertension treatment or adjusted for hypertension treatment in our definition of the phenotype. A region with at least two statistically significant markers, such as that found on chromosome 1, may indicate a region in which a candidate gene associated with the rate of change in SBP may lie. This region has not been as heavily studied as those mentioned previously, but is a viable candidate region because of the proximity of the angiotensinogen gene (~244–251 cM) . Two pre8–269 cM.The use of linear mixed models to define phenotypes is a methodology that allows for the adjustment of the main factor by covariates. The phenotypes defined for each subject are continuous and generalizable. Future work should be done in the area of combining this phenotype estimation directly with the linkage analysis so that the error in estimating the phenotype can be properly incorporated into the genetic analysis, which, at present, assumes that the phenotype is measured (or estimated) without error.


An initial linkage analysis of the alcoholism phenotype as defined by DSM-III-R criteria and alcoholism defined by DSM-IV criteria showed many, sometimes striking, inconsistencies. These inconsistencies are greatly reduced by making the definition of alcoholism more specific. We defined new phenotypes combining the alcoholism definitions and the latent variables, defining an individual as affected if that individual is alcoholic under one of the definitions (either DSM-III-R or DSM-IV), and indicated having a symptom defined by one of the latent variables. This was done for each of the two alcoholism definitions and five latent variables, selected from a canonical discriminant analyses indicating they formed significant groupings using the electrophysiological variables. We found that linkage analyses utilizing these latent variables were much more robust and consistent than the linkage results based on DSM-III-R or DSM-IV criteria for definition of alcoholism. We also performed linkage analyses on two first prinicipal components derived phenotypes, one derived from the electrophysiolocical variables, and the other derived from the latent variables. A region on chromosome 2 at 250 cM was found to be linked to both of these derived phenotypes. Further examination of the SNPs in this region identified several haplotypes strongly associated with these derived phenotypes. An often challenging and sometimes underappreciated facet of genetic epidemiology is the process of choosing an appropriate definition of the trait of interest. Consider for example Alzheimer's disease. While advanced age is the main risk factor for this disease, and it most often strikes after age 65, there are people that develop this disease before the age of 50. Pathologically, the disease appears the same regardless of the age of onset: microscopic examination of brain tissue from patients that are struck with this disease at any age reveal the presence of both amyloid plaques and neurofibrillary tangles. However, it is now recognized that the patients with the very early ages of onset have a mutation in one of three genes APP, PS1, PS2), and patients that develop Alzheimer's disease at a later age lack these mutations [, PS2, anThis problem is even more complicated with a behaviorally defined phenotype such as alcoholism, as there is no completely objective laboratory test for the phenotype. In this analysis, we explore the problem of phenotype definition with the Collaborative Study on the Genetics of Alcoholism (COGA) dataset, and look at alternate definitions of the phenotype. Of particular interest is the problem of lack of consistency in results due to a change in the definition of alcoholism from DSM-III-R to DSM-IV criteria .We performed model-free linkage analyses using the program SIBPAL, part of the SAGE analysis package . InitialTwo sets of linkage analyses were performed using two different phenotypes: the phenotype of alcoholism defined by each of the two criteria: DSM-IIIR and DSM-IV. These were coded as dichotomous variables, and denoted respectively as Alc1 and Alc2. The following covariates were used in all linkage analyses: dichotomous indicator of habitual smoking; dichotomous indicator of whether an individual was Black (derived from ethnicity); dichotomous indicator of whether an individual was White (derived from ethnicity); age at interview; square of age at interview; cube of age at interview. The results of the initial linkage analyses are shown in Figure p-values are not valid due to the non-independence of family members, they gave us an approximation of the relationships between the latent variables and the EEG variables. We found that latent variables 1 (persistent desire to stop drinking), 2 (morning drinking), 7 (gave up activities to drink), 9 , and 11  showed evidence of significant grouping with the EEG variables. Motivated by these observations, we defined ten derived phenotypes named Ai_j for i = 1, 2 and j = 1, 2, 7, 9, or 11 as defined above; an individual was affected with derived phenotype Ai_j if they were defined as alcoholic under definition 'i' and also had the symptom defined by latent variable 'j'. Correlations between these phenotypes were all higher than those between the original Alc1 and Alc2 variables (data not shown), and as shown in Figure We then made the assumption that the latent variables and electroencephalogram (EEG) variables are phenotypes that could be used to identify more homogeneous alcoholism phenotypes. We performed a canonical discriminant analysis using each of the latent variables as a potential grouping variable for the EEG variables. Although the To investigate linkage for phenotypes derived from the EEG variables and the latent variables, we formed several phenotypes using principal components analysis to summarize collections of variables. We used the first principal component derived from all 13 EEG variables, and also the first principal component derived from alcoholism and latent variables. The comparison of the linkage results for these two outcomes is given in Figure We chose to follow up on these chromosome 2 results, especially in the area around 250 cM, with further analyses using the Affymetrix SNP data. We examined the evidence for linkage using only the SNP data for all of chromosome 2. The linkage results for the EEG principal component outcome is shown in Figure To attempt to narrow the linkage peaks on 2q, we combined the STR and SNP markers, utilizing a combined map based on the map data sent along with the marker file, which produced the linkage results for our alcohol and latent variable and EEG principal component outcomes shown in Figure We used the program FBAT to examine evidence for association between SNP genotypes and each of the outcome variables of interest. There were a total of ten phenotypes of type Ai_j, and the two principal component derived phenotypes mentioned above . Each association can be tested using either a dominant model for mode of inheritance , or an additive model . Thus, for a given SNP, up to a total of 24 association tests can be significant. Figure We followed up on these results by examining the associations between these outcome variables and haplotypes formed from SNPs in this region using the HBAT function in the program FBAT. We chose haplotypes based on three criteria: 1) if there were consecutive SNPs that all showed a large number of significant results in Figure We have identified several alternative phenotypes defined by use of latent and/or EEG variables that provide consistent linkage signals on 2q using STR markers. We confirmed this signal on 2q using the Affymetrix SNP data. We then combined the SNP and STR data and show that the combined information narrows the region and increases the evidence for a candidate region with susceptibility genes for Alcoholism or related traits.Previous linkage analysis on comorbidity of alcohol dependence and habitual smoking revealed a modest peak on chromosome 2 around 90 cM, while evidence for linkage to our region of interest was weak and inconsistent in that study . Linkagep-values. Application of the conservative Bonferroni correction can be easily applied to these p-values, though this study is hypothesis generating rather than hypothesis testing, so we felt this unnecessary. Our results mirror the linkage signal previously found utilizing EEG phenotypes reported by Porjesz et al. [Investigating multi-SNP haplotype associations, we find that there are three SNP haplotypes showing consistent and significant associations at positions ~206, 210, and 228 cM on chromosome 2q. However, the principal component derived phenotypes were not found to show significant haplotype associations as those found with the latent variables. However there is a 5-SNP haplotype at 234.0–234.5 that shows significant association with the EEG principal component. We have analyzed multiple phenotypes and multiple SNP haplotypes for these association-based analyses, a total of 24 separate analyses, and have reported here the uncorrected z et al. .COGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramSNP: Single-nucleotide polymorphismSTR: Short tandem repeatHWW participated in the design of the study and performed the statistical analysis. RCPG conceived of the study design, and participated in the study design and coordination, and helped to draft the manuscript. HT, VG, and GP participated in the selection of appropriate analyses and helped to draft the manuscript. All authors read and approved the final manuscript.


We performed variance components linkage analysis in nuclear families from the Framingham Heart Study on nine phenotypes derived from systolic blood pressure (SBP). The phenotypes were the maximum and mean SBP, and SBP at age 40, each analyzed either uncorrected, or corrected using two subsets of epidemiological/clinical factors. Evidence for linkage to chromosome 8p was detected with all phenotypes except the uncorrected maximum SBP, suggesting this region harbors a gene contributing to variation in SBP. Linkage analysis of quantitative traits holds great promise for dissecting the genetic contribution of phenotypes that vary in the population. While only the extreme trait values may be of clinical relevance, the full distribution in the population provides additional power to identify genetic determinants. However, the variation in trait value will depend on the underlying genes, additional environmental factors, and other correlated phenotypes that may also be under genetic control. The underlying trait distribution after removing these factors may have higher power to detect linkage. However, removing variation due to factors which themselves have a genetic basis  may remove genetic variation due to common genes contributing to both phenotypes, or where the genes for the explanatory variable (weight) contribute a major proportion of the genetic variation in the major phenotype of interest (systolic blood pressure).In this study, we analyzed nine phenotypes from the Genetic Analysis Workshop 13 Framingham Heart Study data, based on systolic blood pressure (SBP). We explore how the choice of underlying phenotype  and correction for other variables affects the results of variance components linkage analysis.Three baseline SBP response variables were used in the analysis: mean SBP, maximum SBP, and SBP at age 40 . Mean and maximum SBP were calculated for all study participants in Cohorts 1 and 2; no age limit was imposed and no minimum number of SBP readings were required for an individual to be included in the analysis. For SBP at age 40, the value from the first visit following a participant's fortieth birthday was used, or the final reading if the participant was under age 40 at their final study visit. Any participant who had no SBP recorded between the ages of 35 and 45 years was excluded from the age 40 analysis. It was assumed that all missing data occurred at random and would not bias results.For each of the three response variables, three levels of correction were applied. As well as the unadjusted response (A), adjustments were made for epidemiological and clinical covariates (B) and epidemiological covariates only (C). Adjusted phenotypes were obtained from the residuals after fitting a linear model using SPLUS v5.1. COHORT was included in all adjusted phenotypes, otherwise only those covariates that were statistically significant at the 5% level using a forward model selection process were included. For adjustment B, covariates included in the variable selection process were age, sex, cigarettes per day (SMOKE), units of alcohol per day (DRINK), treatment for high blood pressure (TREATED), and body mass index (BMI). Adjustment C included all covariates except TREATED and BMI. For maximum SBP and SBP at age 40, the corresponding covariate measure was used. When analyzing mean SBP, the covariates SMOKE, DRINK and TREATED were defined as yes or no according to whether these factors has ever occurred. The resulting nine phenotypes will be referred to as MAXSBP, MEANSBP, and AGE40SBP with suffixes A, B and C referring to the three levels of correction Table .Extended pedigrees were split into nuclear families due to the constraints of GENEHUNTER. In total, 294 nuclear families (242 extended pedigrees) from 330 original pedigrees contributed to the analyses, having two or more genotyped sibs with trait values for at least one of the phenotypes studied Table . QuantitGenome-wide linkage analysis results for each of the nine phenotypes are shown in Figure Chromosomes 12 and 22 showed suggestive linkage with MEANSBP_A only; the highest LOD score in these regions from the remaining phenotypes was 1.1 (chromosome 12 AGE40SBP_A) and 1.4 . In total, 14 regions across the genome attained a LOD score > 1.0. Of these, 11 included a phenotype adjusted for epidemiological and clinical covariates (B), compared with two unadjusted (A) and three adjusted for epidemiological covariates only (C). These results are consistent with the higher sib correlation observed for phenotypes B than C  in this region contribute directly to SBP, and not to an intermediate phenotype which has been corrected for . All SBP-based phenotypes showed some evidence for linkage on chromosome 8 (LOD > 1.5), except the unadjusted MAXSBP. Further analysis of SBP corrected for individual epidemiological and clinical factors might identify the crucial factors to be included in the correction for SBP.The maximum LOD score over the nine traits is 2.5. Although multiple testing has been performed, we note that the traits are not independent  and may reduce much of the age-dependent variation in SBP which is present in other unadjusted phenotypes. For example, the maximum SBP is treated similarly for study participants from age 20–30, or from age 40–70, although the distribution of SBP differs substantially in these age ranges. The linkage results suggest that modelling by age and epidemiological or clinical factors may increase the power to detect linkage to SBP.


To assess the factors affecting the incidence of radiation-induced dermatitis in breast cancer patients treated with adjuvant 3 D conformal radiotherapy by the analysis of dosimetry and topical treatments.in vivo.Between September 2002 and July 2009, 158 breast cancer patients were treated with adjuvant 3 D conformal radiotherapy after undergoing surgery. Before November 2006, 90 patients were subjected to therapeutic skin care group and topical corticosteroid therapy was used for acute radiation dermatitis. Thereafter, 68 patients received prophylactic topical therapy from the beginning of radiotherapy. The two groups did not differ significantly in respect of clinical and treatment factors. Furthermore, the possible mechanisms responsible for the effects of topical treatment on radiation-induced dermatitis were investigated 107%; >28.6%) and volume receiving 110% of prescribed dose within treated volume , and no prophylactic topical therapy for irradiated skin, were associated with higher incidence of acute radiation dermatitis. The protective effect of prophylactic topical treatment was more pronounced in patients with TV-V110% > 5.13%. Furthermore, using irradiated mice, we demonstrated that topical steroid cream significantly attenuated irradiation-induced inflammation, causing a decrease in expression of inflammatory cytokines and TGF-beta 1.The incidence of radiation-induced moist desquamation was 23% across 158 patients. Higher volume receiving 107% of prescribed dose within PTV (PTV-V110% > 5.13% may be an important predictor for radiation induced dermatitis. Prophylactic topical treatment for irradiated skin can significantly improve the tolerance of skin to adjuvant radiotherapy, especially for patients with higher TV-V110%.TV-V Radiotherapy (RT) is commonly used as an adjuvant modality in the treatment of breast cancer ,2. AdjuvRT-induced skin toxicity is a prominent clinical problem affecting the majority of breast cancer patients receiving adjuvant RT and can lead to temporary or permanent cessation of treatment. Severe skin reactions may be painful, lead to localized or occasionally systemic infection, and cause permanent scarring. The incidence of RT-related toxicity may be reduced by refinements in radiation techniques, such as improving dose conformity and dose homogeneity within the irradiated area. It is reported that breast IMRT could reduce approximately 15-20% moist desquamation of the irradiated skin by delivering a more homogenous dose of radiation through the breast and efficiently removing the radiation hot spots ,10. Accoin vivo.It is recommended that skin in the irradiated area be kept clean and free from trauma. Many physicians commence topical therapy at the clinical onset of radiation dermatitis but there is no consensus regarding the most appropriate timing or agents for topical therapy in such instances. Recently, some agents including creams containing urea and steroid, have been investigated and showed significant effects to reduce radiation induced dermatitis -13. HoweThis retrospective study was approved by the Institutional Review Board, and a waiver of informed consent was obtained. The patient data consisted of women who had undergone surgery for breast cancer followed by adjuvant 3D-conformal radiotherapy in our department between September 2002 and July 2009. Patients treated before November 2006 did not receive topical therapy for the skin until the onset of radiation dermatitis (therapeutic skin care group). Patients treated after this date underwent prophylactic topical therapy, including steroid cream (0.1% mometasone) and barrier film spray (3M™Cavilon No-sting Barrier Film) (prophylactic skin care group). Prophylactic medication was applied to the treatment field every three days from the start of RT . In gene107% (percent volume receiving 107% of prescribed dose within PTV) and TV-V110% (percent volume receiving 110% of prescribed dose within treated volume (TV)) to identify the hot spot area within and outside the target. The definition of treated volume is that volume enclosed within the prescribed dose, and the areas receiving excessive dose, especially >10% of prescribed dose, are known as radiation hot spot. Figure 107% and TV-V110% related to the location of moist desquamation.Radiotherapy was planned using the Eclipse Planning System , and treatment was delivered using Varian 21EX. All patients were treated with 3 D conformal radiotherapy. For radiation therapy with 3D-CRT, a customized immobilization device was developed which encompassed the upper extremities, head, neck and chest, to minimize variability in the daily setup. The clinical target volume (CTV) was contoured on the individual axial CT slices with 5 mm slice thickness of each patient. The CTV was expanded by 10 mm, but within 3 mm of the skin surface, to create the planned target volume (PTV). Treatment plans were developed by applying tangential photon fields set up isocentrically, with or without individually weighted segmental fields superimposed on the tangential fields and 1-2 coplanar, different gantry angle fields. Wedges were used in almost all cases. Our planning goals were to provide a homogenous PTV dose of 50.4 Gy, while minimizing the dose delivered to the lung, heart and contralateral breast. Furthermore, to evaluate the effects of dose inhomogeneity on acute skin toxicity, we analyzed several dosimetric factors including the planning target volume (PTV), PTV-V2 test was utilized to compare acute skin toxicity between different sample groups, and to analyze associations between acute toxicity, dosimetric parameters and clinical characteristics. Statistical significance was assumed at p < 0.05.The χThirty male BALB/c mice, aged between 8 and 10 weeks old, were purchased from the National Science Council, Taiwan. Protocols relating to animal experimentation were approved by the Chang Gung Memorial Hospital Laboratory Animal Center. For irradiation experiments, anesthetized mice restrained in modified Perspex tubes and covered with a 0.3 cm bolus, received a irradiation dose of 15 Gy in a single fraction to the skin by 6 MeV electron from a linear accelerator . UnirradAt the indicated times after irradiation, three mice from each group were sacrificed by cervical dislocation, and irradiated skin was dissected and stored at -80°C pending analysis . SpecifiEqual amounts of protein were loaded on to SDS-PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were incubated with antibodies specific for TGF-β1, IL-6, MCP-1 and COX-2 , followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected using enhanced chemiluminescence. To normalize protein loading, the membrane was re-probed with mouse anti-r-tubulin antibody (1:1000).Cellular aspects of inflammation were measured in skin tissue samples using immunochemical staining. Experimental and control mice were sacrificed by cervical dislocation 20 days after exposure to 15 Gy irradiation. The tissues were fixed in 10% buffered formalin, paraffin-embedded and sectioned at an average thickness of 5 μm. Briefly, samples were incubated overnight with goat anti-mouse TGF-β1 antibody  diluted 1:20 in 0.01 M RPMI at room temperature. After washing three times with PBS, the sections were incubated with biotinylated anti-goat IgG (1:100) for 10 min followed by peroxidase-avidin staining. Samples were washed with PBS, followed by addition of 3-amino-9 ethylcarbazole.107% and TV-V110% within the 158 patients were 28.6% and 5.13% respectively. In comparison of dosimetric parameters between the 2 groups, the mean dose, target coverage and dose inhomogeneity did not differ significantly between patients receiving chest wall irradiation or whole breast irradiation (Table 107% and TV-V110%) and surgery type did not differ significantly between the two groups, but the incidence of moist desquamation was significantly greater in the therapeutic skin care group than in the prophylactic skin care group . Furthermore, within the prophylactic skin care group there was no statistical difference in the incidence of radiation dermatitis between those patients treated with topical steroid cream and those treated with barrier film spray .One hundred and fifty-eight patients met the study criteria and completed the planned course of treatment. Ninety patients underwent chest wall irradiation and 68 patients received whole breast irradiation. The mean age (±SD) of the overall study population was 50 ± 11 range 24-86) years. The incidence of moist desquamation was 23% of the total study population, 26% for patients who underwent chest wall irradiation and 19% for those that received whole breast irradiation. Among 37 patients developed moist desquamation,34 patients appeared grade 2 and 3 had grade 3 radiation dermatitis. The details of the treatment parameters are listed in Table 4-86 year107% (>28.6%), higher TV-V110% (> 5.13%) and no prophylactic topical therapy for irradiated skin, were significantly associated with moist desquamation  and group II (comprising 79 patients with TV-V110% > 5.13%), and further analyzed the risk factors associated with higher grade skin toxicity for each group. We found no significant association between the incidence of acute skin toxicity and surgery type, PTV-V107% and prophylactic skin care or not for group I (Table 110% > 5.13%), prophylactic topical therapy significantly decreased the incidence of higher grade skin toxicity (p=0.008)  Table .Real-time RT-PCR was utilized to quantify the expression of cytokines induced by radiation and changes in expression after topical treatment. Low expression levels were noted in unirradiated control mice and there were no significant changes in expression after topical treatment (unirradiated F-mice and S-mice). Irradiation (15 Gy) induced a significant increase in the mRNA levels of the cytokines detected in the experimental groups compared with the unirradiated group after 24 h. Treatment with steroid cream significantly attenuated the increase of pro-inflammatory cytokines in cutaneous tissues; barrier film spray had no effect Figure . WesternTGF-β1 has been reported as an important predictive biological marker for RT-induced inflammation and fibrosis . TherefoPostoperative radiotherapy has become an integral part of the complex treatment of breast cancer. The risk of acute and late RT-induced sequelae increases with radiation exposure of the organ at risk. The development of appropriate methods for preventing and treating established radiation-induced skin toxicity would be helpful for approximately 25% of breast cancer patients who develop moist desquamation or ulceration of the irradiated chest wall/breast skin ,10. At p107%) and > 55.4 Gy within treated volume (TV-V110%) were significant predictors of RT- induced skin toxicity. The similar percentages of patients with higher PTV-V107% and TV-V110% may explain why the mode of surgery did not significantly affect the incidence of radiation dermatitis.In recent years, 3D-CRT, IMRT and tomotherapy have become promising treatments to improve conformality and dose homogeneity for adjuvant RT treatment of breast cancer. However, there have been few studies concerning the correlation between dosimetric parameters and the incidence of radiation-induced skin toxicity. In the present study, we demonstrated that a larger volume receiving >53.9 Gy within PTV  provided significant protection from radiation-induced dermatitis. There are still no well established prophylactic treatments to prevent radiation skin toxicity despite some agents are reported to reduce radiation induced dermatitis, including topical vitamin C, creams containing urea and steroid -13,17-19in vivo is associated with increased TGF-β1 and COX-2 expression, and inflammatory cytokines. Therefore, we assessed the potential of treatment with topical steroid cream to mitigate skin toxicity caused by irradiation in animal studies. Topical steroid cream decreased the RT-induced inflammatory response, causing a reduction in levels of the pro-inflammatory cytokines IL-1α/β, TNF-α, TGF-ß1, IL-6 and MCP-1. Prophylactic barrier film spray had no effect on the production of early inflammatory cytokines after radiation exposure in mice. However, analysis of inflammatory cytokine RNA, TGF-β1 and COX-2 protein expression one week after irradiation, and TGF-β1immunochemical staining 20 days after irradiation, demonstrated that both topical steroid and barrier film spray did have an impact on the mitigation of radiation dermatitis. Based on our clinical data and experiments in vivo, we suggest that prophylactic topical steroid treatment inhibits RT-induced inflammation, leading to decreased radiation dermatitis. The mechanism responsible to barrier film spray- induced decrease in radiation dermatitis might be to decrease skin trauma and skin irritation. However, the effects of barrier film spay on irradiated skin and the underlying mechanisms still need further investigation.Clinically, cutaneous inflammation after irradiation of normal tissue can lead to both temporary and persistent complications. In mice, early radiation dermatitis usually peaks at 20 days . RT-indu107% and TV-V110% have a significant impact on radiation-induced dermatitis. By multivariate analysis, TV-V110% > 5.13% is an important predictor of the incidence of moist desquamation. In addition, prophylactic topical treatment for irradiated skin could significantly improve the tolerance of skin to adjuvant radiotherapy, especially for patients with higher TV-V110%. However, due to the limitation of retrospective study, a larger study and a randomized trial are needed to confirm these findings.Our results suggest that dose inhomogeneity as measured by PTV-VThere is no conflict of interest that could be perceived as prejudicing the impartiality of the research reportedMFC conceived of the study, performed the study, drafted the manuscript and participated in coordination. WCC participated in coordination and assisted in editing of manuscript. CHL helped in the collection and analysis of clinical data. CHH and KCL performed statistical analysis and participated in its design YHC helped in the collection of clinical data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/508/prepub


Intestinal atresia is a rare congenital disorder with an incidence of 3/10 000 birth. About one-third of patients have severe intestinal dysfunction after surgical repair. We examined whether prenatal gastrointestinal obstruction might effect on the myenteric plexus and account for subsequent functional disorders.We studied a rat model of surgically induced antenatal atresia, comparing intestinal samples from both sides of the obstruction and with healthy rat pups controls. Whole-mount preparations of the myenteric plexus were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (nNOS). Quantitative reverse transcription PCR was used to analyze mRNAs for inflammatory markers. Functional motility and permeability analyses were performed in vitro. Phenotypic studies were also performed in 8 newborns with intestinal atresia. In the experimental model, the proportion of nNOS-immunoreactive neurons was similar in proximal and distal segments , but proximal segments contained a higher proportion of ChAT-immunoreactive neurons . Phenotypic changes were associated with a 100-fold lower concentration-dependent contractile response to carbachol and a 1.6-fold higher EFS-induced contractile response in proximal compared to distal segments. Transcellular (p = 0.002) but not paracellular permeability was increased. Comparison with controls showed that modifications involved not only proximal but also distal segments. Phenotypic studies in human atresia confirmed the changes in ChAT expression.Experimental atresia in fetal rat induces differential myenteric plexus phenotypical as well as functional changes (motility and permeability) between the two sides of the obstruction. Delineating these changes might help to identify markers predictive of motility dysfunction and to define guidelines for post-surgical care. Intestinal atresia is a common congenital gut disorder characterized by the interruption of intestinal continuity. Its prevalence is about 3/10 000 births (1). The diagnosis is generally made during the second or third trimester, based on ultrasound detection of bowel dilation Initiation and regulation of small-bowel motility depends on normal functioning of several structures, and particularly the enteric nervous system (ENS). The ENS is an integrative network made up of neurons and glial cells derived from the neural crest. It is located all along the gut and regulates intestinal peristalsis and secretion Although it is well recognized that atresia leads to morphological alterations in proximal and distal segments, there is so far little evidence that ENS alterations participate in the motility disorders observed after surgical treatment of congenital intestinal atresia. Only scattered and descriptive assessments of the ENS are available. Current data points largely to alterations in the proximal dilated segment but fail to show major alterations in the distal segment. In particular, decreases in NADPH-diaphorase, PGP9.5 and VIP nerve density have been described upstream of the atresia In the present study, using an experimental rat model of prenatal intestinal atresia Only rat fetuses with successful prenatal obstruction (n = 44) were included in the study. Birth weight was 4.5±0.5 g and total small bowel length was 12.8±1.3 cm. Littermate birth weight was 5.3±0.4 g  and intestinal length 15.3±1.8 cm .The atresia was located at a median of 8 cm (range 2–13 cm) from the duodeno-jejunal junction. The external diameter of the gut was significantly larger (1.8±0.4-fold) in the segments proximal to the atresia than in the distal segments . For eac2)) was significantly lower in the proximal segments than in the distal segments . There was no significant difference in the density of SOX10-IR glial cells  and controls samples (n = 8). Carbachol induced a concentration-dependent contractile response in proximal segments, starting at a concentration of 1 microM . This reThe neurally mediated contractile response of small-intestine longitudinal muscle was also assessed ex vivo in organ chambers in atresia samples (n = 12). EFS-induced contractile responses were 1.6-fold higher in proximal than distal atresia segments  did not significantly modify EFS-induced contractile responses in neither proximal nor distal segments  and distal segments (16.9±5.1 ohms.cm−2) or between atresia segments and control jejunum (18.2±3.5 ohms. cm−2) or control ileum (16.8±7.3 ohms. cm−2).Tissue resistance was not significantly different between proximal  and ileal segments  of controls  flux was significantly higher in the proximal segments than in the distal segments , 2.7-fold higher for Il6 (p = 0.01), 1.7-fold higher for Il10 (p = 0.01) and 1.6-fold higher for Il1 beta (p = 0.03). In contrast, iNos and Ifn gamma mRNA expressions were similar in the two segments (p = 0.77 and p = 0.06 respectively).The human atresia (N = 8) was located at a median of 51 cm (range 3–125 cm) from the duodeno-jejunal junction. The external diameters ranged from 11 to 25 mm for the proximal segments and from 4 to 6 mm for the distal segments. The ratio of the diameters was 3.6±1.3.H&S staining showed a normal mucosa in all cases, with submucosal edema in five cases and mucosal hemorrhagic spots in three cases. In all cases, muscular hypertrophy was present in the proximal segments when compared to the distal segments, with a muscle thickness ratio of 1.4 to 2.2 . As prevIn human atresia, the proportion of ChAT-IR myenteric neurons was higher in the proximal (38±16%) than distal segments (25±12%), although the difference did not reach statistical significance , IL1 beta (p = 0.7), IL6 (p = 0.9), IL-10 (p = 0.8), iNOS (p = 0.8) or IFN gamma (p = 0.4) was observed (data not shown).The experimental protocol was approved by the French animal care and use committee  and all animal procedures were performed according to the French guidelines for animal protection and animal welfare.According to the guidelines of French Ethics Committee for research on human tissues and after Institutional Review Board CPP Necker approval, atresia specimens were considered as “residual tissues”. Parents’ written informed consent was obtained for all the patients and the tissue collection was declared to the Research Ministry N° DC-2009-955.Time-dated pregnant rats were purchased from Janvier Laboratories . The surgical procedure was performed on day 18 of gestation (term: 22 days±1), as previously described Caesarean delivery was performed on day 21 of gestation. The fetuses were killed according to the guidelines of the animal care and use committee and the whole intestine was removed. Atresia was considered successful if the fetus was alive at delivery and if a stricture of the small intestinal was clearly visible, together with a difference in gut diameter above and below the ligation, and a difference in the color of the two segments .We compared two segments from each animal; one located 1 cm immediately above the atresia  and the other 1 cm immediately below it . In addition, segments of intestine from rat pups obtained after caesarian delivery at day 21 were collected and processed similarly to atresia segments. Two different parts were studied: jejunal samples, located at one cm below the duodeno-pancreatic zone and ileal samples, located at one cm before the ileocaecal valve.We studied eight neonates (6 girls and 2 boys) born with isolated intestinal atresia with a median gestational age of 36 weeks (range 32–39) and mean birth weight of 2660±540 g. In keeping with French ethical guidelines on research involving human tissues, intestinal fragments resected during curative surgery at birth were considered as residual tissue. Intestinal dilation had been diagnosed prenatally in all but one of the children, at a median of 27 weeks of gestation (range 22–33). None of the patients had short bowel syndrome but all showed post-operative intestinal dysmotility with a median duration of parenteral nutrition of 36 days (range 22–136 days). The median length of hospital stay was 54 days (range 24–141 days).Because of the small size of the human atresia distal segment samples, ex vivo functional studies could not be performed and the priority for IHC staining and PCR analysis was given.3 containing 0.5% Triton X-100 and 5% horse serum (Sigma), whole-mount preparations of rat and human atresia were incubated overnight with anti-choline acetyltransferase, anti-neuronal nitric oxide synthase and anti-Hu antibodies. Anti-Sox10 and anti-Caspase3 antibodies were only used for rat specimens. Glial cells were counted after Sox10 labeling. Apoptosis was analyzed after Caspase3 staining. The tissue samples were then washed with PBS (3×10 min) and incubated for 12 h with the following secondary antibodies. Antibody sources and concentrations are shown in Intestinal samples were fixed overnight at 4°C in 0.1 mol/l PBS containing 4% paraformaldehyde. Whole mounts of the longitudinal muscle myenteric plexus were prepared by microdissection. After permeabilisation for 3 h in PBS/NaN2), as previously described 2 unit.Specimens were examined with LSM 510 META confocal fluorescence microscope equipped with a 40X objective . The proportion of ChAT-immunoreactive (IR) and nNOS-IR neurons was normalized to the total number of Hu-IR neurons. Contiguous non-overlapping fields were analyzed until 500 Hu-IR neurons had been counted. The packing densities of glia and neurons were also evaluated and expressed as a function of tissue area  according to the manufacturer’s instructions, and transcripts were analyzed with Taqman assays (Applied Biosystem) on a 7300 Applied Biosystems device. Cytokine  were normalized to S6. ChAT and nNos transcripts were normalized to Pgp9.5. Cytokine Results were analyzed with the delta Ct method. Primer sources are shown in 2, 95% O2) Krebs-bicarbonate solution , and allowed to equilibrate for 30 minutes. Isometric contractions were recorded with a force transducer . Electrical field stimulation (EFS) was applied with a stimulator connected to platinum electrodes in order to activate enteric neurons and to characterize neurally mediated contractile responses. The parameters of stimulation were as follows: 12 V, 20 Hz, train duration 10 s and stimulus pulse duration 300 micros. The results were normalized to tissue-wet weight and expressed in N/g.The contractile activity of intestinal segments was analyzed as previously described −6 mol/l; Sigma) and a nitric oxide synthase inhibitor . Atropine and L-NAME were applied 15 minutes before EFS. At the end of the experiments, after abundant washing for 30 minutes, increasing concentrations of the cholinergic agonist carbachol chloride  were added to evaluate contractile responses.The neuropharmacology of EFS-induced contractile responses was analyzed in the presence of a muscarinic antagonist  was added to the luminal buffer 15 min after sample mounting. After 2 hours, the enzymatic activity of intact HRP was measured with a modified Worthington method.Horseradish peroxidase  was used to analyze changes in transcellular permeability. Type VI HRP  was used to analyze changes in paracellular permeability. 14C-mannitol was added to the mucosal side. After 2 hours, 14C-mannitol fluxes  were analyzed with a liquid scintillation counter .Non-parametric tests were used. Results for proximal and distal segments were compared by using Wilcoxon’s paired two-tailed test or the Kruskal-Wallis test. Differences were considered significant at p<0.05.In our experimental model of prenatal intestinal obstruction, prenatal mechanical constraints resulted in major phenotypic and functional differences between the myenteric plexus immediately proximal and distal to the obstacle, whereas these two adjacent segments were expected to be similar. These changes were characterized by a higher proportion of cholinergic neurons and an increased cholinergic neuromuscular contractile response in the proximal segments as compared to the distal segments.The rat model of prenatal obstruction was developed to determine whether and how prenatal atresia might affect the functional and/or phenotypic properties of the upstream and downstream enteric nervous system (ENS). In this model, complete intestinal obstruction is induced prenatally, resulting in dilation of the upstream segment and narrowing of the downstream segment, as observed in other experimental models and in human prenatal atresia A major finding in our study is the identification of neuroplastic changes in the ENS between the segments immediately upstream and downstream of the atresia. We observed a significant reduction in neuronal density in the proximal segments. This difference could result from tissue distension induced by the obstruction. This mechanism has been suggested in an adult rat model of occlusion, where a lower frequency of nerve fibers in the hypertrophic proximal segment was also observed Besides changes in neuronal density, we observed a higher proportion of ChAT-IR neurons in the proximal segments, as compared to the distal segments. As post-natal maturation of the ENS has been associated with an age-dependent increase in the expression of the vesicular acetylcholine transporter and ChAT in myenteric neurons Postnatal maturation of ENS has been associated with increased in neuronal surface Intestinal motor responses to carbachol also differed between the two segments of atresia. In particular, the proximal segments exhibited a stronger contractile response to carbachol as compared to the distal one. Furthermore, carbachol induced dose-responses in the proximal and the distal segment were similar to the one observed in the jejunum and ileum of healthy controls, respectively. This further reinforces the hypothesis of a defect in functional maturity of the distal segment. In controls, the lower contractile response induced by carbachol observed in the ileum vs. jejunum is consistent with the rostro caudal maturation gradient of the gut motor activity previously reported Concerning epithelial barrier function, transcellular permeability to HRP was significantly higher in the proximal segment than distal, and was also higher than in segments from controls. The mechanism underlying this increased permeability is unclear. IFN gamma has been shown to enhance paracellular and transcellular permeability The mechanisms responsible for the changes observed after prenatal intestinal obstruction are unclear, but they might result in part from a lack of mechanical or nutritional stimulation in the distal segment, that is mandatory for phenotypic and functional maturation. Recent studies have shown that activity-dependent and nutrition-dependent mechanisms can participate in the regulation of neuromediator expression in the ENS in embryonic neurons In order to determine if the observations made in the rat model were consistent with the human disorder, phenotypic studies were performed on 8 intestinal samples from babies with atresia and post-operative intestinal dysmotility . Analysis showed neuronal changes between proximal and distal segments similar to those observed in the experimental model. Because the experimental model reproduces the macroscopic findings observed in human atresia, one might expect similar functional findings in humans, although studies of a larger cohort of patients, including a correlation analysis of intestinal motility, are needed to confirm this important point.In conclusion, our findings point to functional and phenotypical changes in both parts of intestinal atresia, whereas these adjacent segments were expected to be comparable. Reduced ChAT expression by myenteric neurons and a reduced contractile response in the intestinal distal segment of the atresia may contribute to the intestinal motility disorders frequently observed after surgical treatment of congenital atresia. Increased transcellular permeability in proximal segments could also contribute to increased risk of bacterial translocation in these patients. Therefore, analysis of phenotypical and functional abnormalities in neonates with intestinal atresia might help to identify markers predictive of the severity of the alterations and to define guidelines for optimal postoperative management.Table S1Antibody references and concentrations for rat and human immunofluorescence analysis.(DOC)Click here for additional data file.Table S2TaqMan oligonucleotide primers used for rat and human PCR analysis.(DOC)Click here for additional data file.


Within the last few years the field personalized medicine entered the stage. Accompanied with great hopes and expectations it is believed that this field may have the potential to revolutionize medical and clinical care by utilizing genomics information about the individual patients themselves. In this paper, we reconstruct the early footprints of personalized medicine as reflected by information retrieved from PubMed and Google Scholar. That means we are providing a data-driven perspective of this field to estimate its current status and potential problems. Human Genome Project (Lander et al., personalized medicine because it aims to integrate genomics and clinical data from individual patients in order to improve patient care by identifying more efficient treatment strategies (Auffray et al., The In this paper we present a data-driven overview of the early history of personalized medicine. We are using information retrieved from PubMed and Google Scholar to obtain quantitative information statistics of published articles and the community structure of scientists that share a common interest in this topic. From this information we try to identify the potential impact personalized medicine has made so far but also to reveal indicators of problems that might prevent its blossoming. Despite the fact that PubMed and Google Scholar provide a wealth of curated data about various aspects of published articles over the last decades and the research interests of thousands of scholars it should be emphasized that neither has been designed to serve the purpose of our analysis. For this reasons, the drawn conclusions should only be seen as an indicator. Nevertheless, utilizing databases like PubMed and Google Scholar allows to present arguments beyond a mere opinion due to the possibility to compare different aspects quantitatively with each other.ad hoc assumptions that may lead to erroneous interpretations if they would be too far-off the truth.Specifically, to allow for a simplified interpretation of the retrieved numbers from PubMed and Google Scholar we present whenever appropriate a comparison with related topics. For example, rather than presenting an interpretation based on absolute numbers of, e.g., published research papers and review or editorial papers published for personalized medicine we perform a similar analysis for related fields like bioinformatics and systems biology and present a comparative interpretation. This relieves us from the burden to introduce to many personalized medicine we extract information from the literature. Specifically, we are using information provided by PubMed and compare publications in personalized medicine to other fields. Further, we investigate also the content of such publications, as far as this information is provided by PubMed.In order to obtain a general overview of the current state of the field FLink provided by the NCBI. FLink allows to perform a conventional PubMed search with the additional feature that the results can be downloaded as a csv file. The obtained csv files are than parsed by scripts we wrote in the statistical programming language R. The advantage of this approach is that we have convenient access to the same information that is visually represented by a conventional PubMed search but can process this information as desired. For the following analysis we retrieved all data in November 2012.For our following analysis we used the tool We start by showing in Figure Next, we try to get an insight into the distribution of research papers and non-research papers from the listed PubMed publications. Specifically, we define as “non-research” papers review, comment and editorial articles and as research papers we define all remaining articles. Figure The red proportion in each bar corresponds to the percentage of publications that are non-research papers, whereas the blue proportion corresponds to all other publication types. From a comparison of the results one finds that in translational research and in personalized medicine there is a larger percentage of publications devoted to strategic and conceptual questions as represented by review articles, comments or editorial papers and a smaller proportion of publications corresponds actually to original research papers. This could be a reflection of the novelty of these fields and the hopes that accompanies them. For this reason there may be a higher proportion of articles that try to pave the way for these fields, instead of actually contributing new results either from experimental or computational work.In general, personalized medicine has been conceived as a way to improve medical and clinical patient care by taking into account patient specific data. Hence, personalized medicine is inherently connected to a clinical application. In Figure It is somewhat surprising to see that the terms “biomarkers” and “computational” are not frequently present either. This is surprising because personalized medicine is data-driven and these data are analyzed by computational methods aiming to identify important signatures, e.g., in form of biomarkers. A possible reason for the underrepresentation of these terms could be again given by the novelty of this field, as discussed for Figure In order to obtain a general overview of the used genomics data types that occur in any publication type, including original research, review and editorial articles, related to personalized medicine, we use again PubMed and search for conjugated hits, i.e., “personalized medicine” and “term,” whereas the queries for “term” correspond to the x-axis in Figure It is not surprising that “sequencing” surmounts all other terms because it is the prototype technology to obtain genetic information about various aspects of the DNA (Mardis et al., Due to the multidisciplinarity of personalized medicine it would be interesting to know what other interests people have that are interested in personalized medicine. In order to answer this question we are using Google Scholar.In Figure Figure It is without a doubt that the principle idea behind personalized medicine holds a great potential for translational medicine by improving diagnostic, prognostic and therapeutic approaches for patient care. However, besides the seminal work of Chen et al.  only relA principal hurdle for a wider and active engagement of the community in this research area is certainly provided by the still considerable costs incurred by the needed patient-based high-throughput experiments. This is despite the steadily declining costs for DNA sequencing. More important, I think that the major problem is a lack of a precise definition of personalized medicine that would allow an efficient experimental design translating the paradigm into practice.Overall, based on the information we retrieved from PubMed and Google Scholar we think that it is fair to say that personalized medicine is still at the very beginning. It is increasingly recognized within the literature, but a wider impact hasn't been achieved yet.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The prevalence of chronic diseases such as cancer, type 2 diabetes, metabolic syndrome (MetS), and cardiovascular disease increases with age in all populations. Epigenetic features are hypothesized to play important roles in the pathophysiology of age-associated diseases, but a map of these markers is lacking. We searched for genome-wide age-associated methylation signatures in peripheral blood of individuals at high risks for MetS by profiling 485,000 CpG sites in 192 individuals of Northern European ancestry using the Illumina HM450 array. Subjects (ages 6–85 years) were part of seven extended families, and 73% of adults and 32% of children were overweight or obese.Pα=0.05 = 3.65 × 10−7 after correction for multiple testing) of which 14,155 are positively associated with age while 7,967 are negatively associated. By applying a positional density-based clustering algorithm, we generated a map of epigenetic ‘hot-spots’ of age-associated genomic segments, which include 290 age-associated differentially methylated CpG clusters (aDMCs), of which 207 are positively associated with age. Gene/pathway enrichment analyses were performed on these clusters using FatiGO. Genes localized to both the positively (n = 241) and negatively (n = 16) age-associated clusters are significantly enriched in specific KEGG pathways and GO terms. The most significantly enriched pathways are the hedgehog signaling pathway (adjusted P = 3.96 × 10−3) and maturity-onset diabetes of the young (MODY) (adjusted P = 6.26 × 10−3) in the positive aDMCs and type I diabetes mellitus (adjusted P = 3.69 × 10−7) in the negative aDMCs. We also identified several epigenetic loci whose age-associated change rates differ between subjects diagnosed with MetS and those without.We found 22,122 genome-wide significant age-associated CpG sites  contains supplementary material, which is available to authorized users. Chronic diseases such as cancer, type 2 diabetes (T2D), metabolic syndrome (MetS), cardiovascular disease, and dementia constitute the most common health problems seen in developed societies , and their prevalence increases with age in all populations -4. It isEpigenetic mechanisms mediate the interaction between gene and environment throughout the lifespan; while the underlying genetic sequence does not change, environmental influences can alter epigenetic marks and thus alter gene expression and induce long-term changes in phenotype and disease susceptibility . The grain utero but these profiles gradually diverge as they get older [DNA methylation is one of the most extensively studied epigenetic mechanisms and plays an important role in the process of development and differentiation . There iet older -38. Seveet older ,39-41.These age-related methylation changes may play a role in the observed age-related risk of various chronic diseases. For example, studies show that the hyper-methylation of certain CpG loci is associated with increased cancer risk via reduced expression of cancer-suppressor genes ,42. It hStudies of genome-wide DNA methylation can be conducted using various populations, and each design has its advantages and disadvantages. For example, monozygotic twins are genetically identical, so epigenetic differences found in twin pairs are likely to be either stochastic or environmentally induced, rather than genetically inherited . In studWhile no epigenome-wide study of extended, multi-generational families has yet been published, a recent study on a combination of twin and their nuclear family members examined the role of genetic features on DNA methylation . These aWe assembled a cohort comprising several large extended families of Northern European descent that is enriched for obesity, central adiposity, and obesity-associated MetS traits. To identify genomic regions whose methylation status changes with aging, we conducted a genome-wide survey of peripheral blood DNA methylation and interrogated more than 485,000 CpG sites in 192 subjects from seven extended families living in two US Midwestern states, Wisconsin and Illinois.The TOPS Family Study of Epigenetics (TFSE) was designed to study the role of epigenetic mechanisms in linking genes and the environment using related subjects of large extended pedigrees. The average age of the cohort is 36.2 ±18.8) years, 28% of the subjects were 18 years and younger at ascertainment and 55% are females. As the subjects were selected from families that are part of a previous genetic study on the metabolic risk complications of obesity , the coh8.8 yearsWe have implemented a data cleaning procedure aiming to retain only the informative CpG probes for downstream analyses . The characteristics of these age-associated CpG sites are shown in Figures M value per year of age in Figure LEP [OLFM4 [IRS2 [TFAP2B [Using methylation status represented by see the ‘’ sectionsee the ‘’ sectiongure LEP , the chiP [OLFM4 , the T2DM4 [IRS2 , and the [TFAP2B   makes them susceptible to developing obesity and MetS, age-associated DNA methylation is enriched in genes and pathways involved in metabolic homeostasis. Changes caused by aging in these epigenetic states might lead to the malfunctions that underlie the increased prevalence of clinical symptoms of obesity and MetS in the aging population ,3. As thLEP, POMC, PPARG, and CNR1, as well as previous obesity GWAS candidates with unclear roles in obesity etiology, such as SIM1, IRX3, and SLC6A11. Furthermore, four genes identified in GWAS for childhood obesity, such as SDCCAG8, TNKS/MSRA, OLFM4, and HOXB5, were found to be epigenetically age-associated as well.We found that 20 genes out of the 36 listed in a gene list based on genetic studies of human obesity  have onePPARG, HNF1B(TCF2), TCF7L2, IGF2BP2, HHEX/IDE , KCNQ1, MTNR1B, ADAMTS9, THADA, and JAZF1. In one of the most recent studies of T2D genes using genome-wide trans-ancestry meta-analysis, seven novel loci were identified [SSR1/RREB1 and LPP. We also looked for any age-associated epigenetic evidence for genes established through approaches other than GWAS and found that the following known T2D genes are under epigenetic regulation by age: IRS1 [IRS2 [AKT [ABCC8 [HNF4A [IPF-1(PDX1) [NeuroD1 [GCK [Of the 20 T2D susceptibility loci recently identified in GWAS , ten werentified . Of thesge: IRS1 , IRS2 [5S1 [IRS2 , AKT [61T [ABCC8 , HNF4A [8 [HNF4A , IPF-1(P-1(PDX1) , NeuroD1[NeuroD1 , and GCKoD1 [GCK .GRB14, KIAA0754, MACF1, MLXIPL, SKIV2L, STK19, TFAP2B, and TRIB1.Of the 25 genes that have been shown to play pleiotropic roles in MetS and inflammation , we founIRS2) on chromosome 13. This cluster begins within the promoter region ), 5′ untranslated region (UTR) and ends in the first exon . A total of 387 GO terms for biological processes were significantly enriched within the aDMC genes , the methylation aging rates did not show any significant difference between the two groups of subjects  in our dataset after QC. Of these, 23 CpG loci were genome-wide significantly associated with age in our cohort. We examined the aging rate of each of these 23 epigenetic markers in the two groups of adult subjects that were separated using the ATPIII definition of MetS (24% with MetS) and found that four sites of four different genes showed nominal differentiation between the two groups  in genomic regions densely packed with age-associated CpG sites. We have named these clusters as aDMCs  and Illinois (one family) and are mostly categorized as working and/or middle class families . There are likely to be broadly similar household routines of dietary intake and activity within these large families. We therefore expect that by using these families to study epigenetic changes over age, we reduce systematic differences in external environmental factors such as geographical location and dietary and lifestyle patterns.The probands of our TFSE cohort were recruited based on the presence of at least two obese and one never-obese sibling(s) in each family, thus raising the likelihood of having obesity-prone genetic patterns in these families. We hypothesize that since our subjects have an obesity-prone genetic background and live in an obesity-inducing environment, the genomic regions that are strongly associated with age are also enriched in gene groups and pathways involved in metabolism-related cascades.IRS2 works as a signaling mediator between cytoplasmic receptor kinases and downstream effectors including PI3 Kinase, Akt, and mTOR [IL-4), and other cytokines [IRS2 include fatty liver disease and glucose intolerance, which is a precursor to MetS [To find these loci, we first searched in our list of age-associated differentially methylated CpG sites and clusters for any gene(s) previously characterized for a role in the etiology of obesity, T2D, or MetS and inflammation. We found that the majority of previously established obesity, T2D, or MetS and inflammation genes overlap with one or more of our age-associated differentially methylated clusters Figure , and 6. and mTOR  and is aytokines . Disease to MetS ,81.SIM1) is a helix-loop-helix PAS domain transcription factor. Sim, its homologue in Drosophila, is a key factor in determining the differentiation of central nervous system (CNS) midline cells [SIM1 gene locus has been found to have caused profound early-onset obesity [SIM1 has an aDMC that spans 22,914 bp covering the 5′-untranslated region to exon 8 containing 44 CpG sites that were significantly associated with age including five sites situated in the promoter region (TSS1500 to first exon).Single-minded 1 , suggesting a possible explanation for these genes having more profound effect on clinical phenotypes in children than in later life.Multiple obesity and T2D candidate genes identified by the GWAS approach were found to have age-associated DNA methylation associations in our analysis. These genes include extensively replicated genes such as  obesity . FurtherNKS/MSRA  were firdatasets . InteresSHH), Indian Hh (IHH), and Desert Hh (DHH), with receptor PACH1 and PACH2 that release its inhibition on the membrane receptor Smoothened (SMO). Released SMO then activates a complex signaling cascade, which leads to nuclear translocation of transcription factors of the Gli (GLI) family and the resultant activation or repression of downstream genes [IHH and DHH, Hh receptor genes PACH1 and PACH2, and SMO and the downstream effector genes GLI2 and GLI3, and several other regulators of the Hh pathways including SUFU and PKA  encode transcription factors that regulate β-cell homeostasis and/or insulin production and secretion, and gene GK is a glucokinase that is involved in beta cell sensing of blood glucose levels [Diabetes mellitus, like obesity, is a chronic condition that increases in its prevalence as people age ,3, but te levels . In our TFAP2B) (cg06117072) encodes a transcription factor from the AP-2 family. This gene has been implicated in dietary weight maintenance [GRB14) (cg04926134) encodes an adaptor protein that binds with insulin receptors and insulin-like growth factor receptors that may have an inhibitory effect on insulin signaling and may play important roles in metabolic homeostasis and growth regulation [TRIB1) (cg14683125), a mitogen-activated protein kinase (MAPK) activation modulator, was found to control migration and proliferation of smooth muscle cells [MACF1) (cg22697325) is a member of protein family that form bridges between different cytoskeletal elements [In this study, we demonstrated the first evidence of differential methylation aging in MetS genes in MetS subjects as compared with non-MetS subjects. Our results showed an interesting pattern in which the epigenetic changes over age are slower in MetS subjects, and its directionality is the opposite to that in non-MetS subjects in all four of these identified loci Table . Our resntenance ,103. Grogulation -107. Trile cells  and has le cells -111. Micelements .With the size of our sample, we do not have enough statistical power to detect all existing signals for MetS-specific aging methylation. We expect that with a larger sample size, one can discover many more disease state-dependent epigenetic markers not only for MetS but also for other aging relevant conditions such as obesity, T2D, dementia, and cancer. This may eventually lead to new clinical approaches in screening and diagnosing people with differential ‘epigenetic risks’ for developing diseases as they age.We examined the epigenetic changes associated with aging in DNA obtained from peripheral blood, a tissue type that is routinely used as a surrogate for mapping age-related DNA methylation changes  because We conducted our study in a family cohort of Northern European descent. Generalization of our findings requires validation in distinct cohorts with similar pedigree structures. It will also be a valuable expansion of our cross-sectional study if we can recall some of our subjects to obtain longitudinal data on the epigenetic changes as well as on the functional status of metabolic pathways over time in the same individuals. Due to the scope and the focus of the current study, we have not looked at the associations of particular methylation loci with phenotypes in our subjects as this will be approached in the future. The connection between epigenetic status and gene expression in blood and certain target tissue types also warrants further investigation.Although our mapping utilized one of the array-based epigenetic platforms that gave the highest available genomic site coverage, it has not nearly exhausted the epigenome. A next-generation sequencing-based approach such on methyl-binding domain-isolated genomic sequencing (MiGS), MeDIP-seq, or bisulphite-sequencing  will be We have conducted one of the first genome-wide surveys of age-associated DNA methylation in a family cohort with large extended pedigrees. In families at high risks for developing obesity-related metabolic disorders, we found age-associated genomic loci densely situate near genes that function in the hedgehog signaling pathway and in MODY. These findings suggest a novel mechanism underlying the gradual deleterious effects of multiple genes and their interactions with nutrition over time, which may contribute to obesity and its complications. The results from this study shed light on the relationship between aging and increased prevalence of obesity, T2D, and related abnormalities and thus may lead to novel approaches for early detection and prevention of these health-endangering conditions.The study cohort consists of 192 individuals ranging in age from 6 to 85 years old representing seven families. Of these, 53 subjects were 18 years and younger at ascertainment and 106 are females. Details of recruitment and phenotyping procedures have been described previously ,115. BriP values excluding X and Y chromosomes. No background subtraction or control normalization was applied with GenomeStudio.Genomic DNA was isolated from peripheral blood after an overnight fast on the same day when each subject was assayed for obesity and MetS phenotypes; thus, the CpG methylation states profiled from these samples reflect the epigenetic status associated with that individual’s current state of body composition and metabolism. One microgram of human genomic DNA was sodium bisulfite-treated for cytosine (C) to thymine (T) conversion using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer’s guidelines. The converted DNA was purified and prepped for analysis on the Illumina HumanMethylation450 microarray following the manufacturer’s guidelines. The Illumina HumanMethylation450k microarray measures the methylation levels of more than 485,000 methylation sites. It includes CpG sites surrounding the transcription start sites  for 99% of RefSeq genes, CpG sites within non-coding RNAs, intergenic regions identified in genome-wide association studies as well as CpG islands/shores/shelves and open sea of the genome. CpG annotations  were identified using the Illumina manifest 1v2.GenomeStudio software and Methylation Module (Illumina) was used to generate final reports containing signal intensities and detection P values of 22 autosomal chromosomes were extracted from GenomeStudio and loaded into Lumi. Next, quality control of the data resulted in the removal of CpG sites with detection P value ≥ 0.01 in more than 5% of the samples . All samples had at least 99% CpG sites with detection P value ≥ 0.01; thus no samples were removed. Recently, multiple groups have reported that this array contains cross-reacting probes that cannot be distinguished between multiple chromosomal positions and that therefore need to be excluded from downstream analysis [For initial quality control preparation of the Infinium Human Methylation 450 K data, we used the Lumi: QN + BMIQ pipeline described previously . Raw siganalysis . Furtheranalysis . Consideβ’ values are defined as follows:Color bias adjustment (Col.Adj) and quantilenormilzation (QN) were performed on signal intensities as implemented in Lumi. Briefly, the QN works on total signal intensity, assuming that the distributions of the pooled methylated and unmethylated probes are similar for different samples. Intensities were then used to generate Beta values. Within Lumi, ‘IM and IU represent the fluorescence intensity originating from methylated or unmethylated CpG locus and α is a constant. Beta mixture quantile dilation (BMIQ) was then performed on β values of QNed data to account for probe type bias. As the Illumina platforms have been shown to discriminate beta values that differ as little as 17% [β values  to ensure probes analyzed might exert a significant biological change. After these steps, a total of 137,168 CpG sites for all 192 samples were imported into data analysis. BMIQ’ed β values for probes with ≥0.17 variation were then converted to M values for data analysis. Lumi defines M values as:where e as 17% ,121, we M values, which are more statistically valid for analysis of differential methylation levels owing to its more homoscedastic nature [All analyses were run using c nature .To estimate cell-type proportions, we used the R minfi package and estimateCellCounts function ,124. ThiDDO: the forward primer, TGTTTAGGAGAAAGGAGTAAGTGATT; the reverse biotinylated primer, ACCCATTATTCACCATACCTACAA; and the pyrosequencing primer, TTTTATGGAGTTGTTTTTGTTAAG. Sepharose beads containing the PCR product were washed and purified using 0.2 M NaOH and the Pyrosequencing Vacuum Prep Tool (QIAGEN). Five microliters of the PCR products was sequenced, and methylation was quantified using the provided software (QIAGEN).DNA methylation at selected sites was validated in a subset of the original cohort by the bisulfite pyrosequencing. This subset consisted of 47 male subjects ages 6 to 21. One microgram of human genomic DNA was sodium bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer’s guidelines. Pyrosequencing was performed using the PyroMark MD system  according to the manufacturer’s protocol. Briefly, the PCR was performed with 10 μM primers, one of which was biotinylated for later purification by Streptavidin Sepharose (VWR). The oligonucleotide primers were purchased from IDT and used for the amplified region of i, the value of a trait Y is modeled as:The quantitative genetic analyses program SOLAR  was usedμ is the trait mean, Xi is a vector of fixed effects measured on individual i, β is a corresponding vector of regression coefficients, and gi and εi are, respectively, a random additive genetic effect and an error term. The covariance of the trait in any two individuals i, j is decomposed as:where ϕi,j is a kinship coefficient  and σ2g and σ2ε are, respectively, additive genetic and residual components of variance. Inclusion of the random effect terms appropriately conditions the estimates of the fixed effect parameters on the relatedness of study subjects. Analyses were performed for each CpG site separately, using M values, where M was modeled as a linear function of age with models that included the random effect of kinship. Sex and cell type composition were included as covariates in all models to account for systematic differences in methylation between men and women. Bonferroni correction for multiple testing, Pα=0.05 = 3.65 × 10−7.where 2A candidate CpG site-based regression analysis against MetS status in each subject was performed to determine if there is differential aging of DNA methylation in subjects with metabolic syndrome compared to those without. In this model, two tests were done: in one, the slopes and intercepts of the regression lines are allowed to differ by MetS status, and in another, a null is forced to be the same. The test of statistically equal intercepts asks whether methylation differs by MetS status for the measured span of ages. The test of equal slopes asks whether MetS impacts change in methylation with age.The R package bump hunter  was usedTo account for array bias, we took the minimum and maximum position of each cluster and looked at the total number of probes from that region that were originally implemented into the analysis. To further define our aDMCs, at least ten CpG sites had to be in the original data set which was implemented into the analysis. At least 50% of those sites had to be genome-wide significantly associated with age. Through this method, we identified a total of 246 aDMCs throughout the autosomal genome. We further looked at the direction of effect age has on each CpG within the identified clusters. If 100% of the CpGs in the cluster had the same direction of effect, it was labeled as ‘positive’ or ‘negative’. If there was variable direction of effects within the cluster, it was labeled as ‘varying’.P value < 0.05 are considered significant.Gene Ontology analysis was done with the FatiGO tool , which u