Quality of life increased significantly in both groups, with no statistical significant difference between groups. We found no evidence for a different treatment effect between patients randomized to home-based AIT compared to patients attending organized rehabilitation . AIT patients reported good adherence to exercise training. Even though these first data indicate positive effects of home-based AIT in patients undergoing coronary artery bypass surgery, more studies are needed to provide supporting evidence for the application of this rehabilitation strategy.Aerobic capacity, measured as the peak oxygen uptake, is a strong predictor of survival in cardiac patients. Aerobic interval training (AIT), walking/running four times four minutes at 85–95% of peak heart rate, has proven to be effective in increasing peak oxygen uptake in coronary heart disease patients. As some patients do not attend organized rehabilitation programs, home-based exercise should be an alternative. We investigated whether AIT could be performed effectively at home, and compared the effects on peak oxygen uptake with that observed after a standard care, four-week residential rehabilitation. Thirty patients undergoing coronary artery bypass surgery were randomized to residential rehabilitation or home-based AIT. At six months follow-up, peak oxygen uptake increased 4.6 (±2.7) and 3.9 (±3.6) mL·kgNCT00363922ClinicalTrials.gov  Our hypothesis was that patients receiving residential rehabilitation would have a higher increase in VO2peak at the follow-up testing compared to the home-based AIT group due to insufficient exercise adherence in the home-based group. A secondary aim of the study was to investigate the feasibility of AIT in a home setting after coronary artery bypass surgery.Cardiac rehabilitation programs including moderate intensity exercise have in meta-analyses been found to prevent mortality in coronary heart disease patients The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. The study was approved by the Regional Committee for Medical and Health Research . Informed, written consent was obtained and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. The trial design was a randomised controlled trail with 1∶1 allocation to parallel groups. Thirty patients undergoing coronary artery bypass surgery  were randomised after surgery to a four week residential rehabilitation program or home-based AIT. Patients were eligible if they went through coronary artery bypass surgery four to eight weeks ago and were clinically stable . Exclusion criteria were left ventricular ejection fraction <30%, contraindications to vigorous physical activity , pulmonary disease clearly limiting exercise capacity, pregnancy, or drug abuse. Data were collected at the Feiring Heart Clinic, Feiring, Norway. The four week residential rehabilitation program was a standard program at the Feiring Heart Clinic rehabilitation centre, and can be regarded as usual care for these patients in Norway. Initially, we had planned to include patients also from another hospital, and to compare residential cardiac rehabilitation with policlinic rehabilitation for the patients recruited at the second hospital. Our sample size estimation was therefore done with regard to also this comparison. With a statistical power of 0.8 (p<0.05) and an expected group difference in improvement in peak oxygen uptake of 3.0 mL/kg/min (±4.0), we estimated that we would have to include 60 patients in total, 30 at each site. As we experienced difficulty in conducting the study at one of the sites, we decided to include only 30 patients to test the hypothesis of the present study.Exercise intensity was set using the Borg 6–20 scale of perceived exertion Patients randomised to home-based AIT received oral instructions in how to do AIT. They were offered 60 minutes of theoretical background for why high intensity training is effective in increasing physical capacity, and practical guidelines in how to do this on their own. They were asked to do the AIT program in the home setting three times per week for a period of six months. In each training session, they should warm up for ten minutes, followed by four intervals of four minutes of high intensity exercise. In these four minutes they should breathe heavily without pain in legs or chest, and with heart rates of 85–95% of individual maximum. After each interval, they should exercise with moderate intensity for three minutes, at heart rate of about 70% of individual maximum. The total exercise time of an AIT session was therefore 38 minutes, of which 16 minutes was high intensity exercise. They were allowed to choose activities that they liked (using large muscle groups), like walking, jogging, swimming or cycling. Regarding safety issues when exercising at home, they were told to contact either the rehabilitation centre staff or their general practitioner if they experienced any symptoms during or after exercising. They got written guidelines describing the AIT program and also about healthy lifestyle, and a training diary. Both groups came back for follow-up testing after six months.2peak) tested on treadmill. Respiratory gas was analysed and a 12-lead electrocardiogram was monitored continuously. We used ramp protocols, individually adjusted to last 8–12 minutes Primary outcome measure was peak oxygen uptake  cholesterol, and triglycerides. Venous blood was drawn after a 10-hour overnight fast. Blood lipids and glucose were analysed using Vitros 350 , ferritin was analysed using Vitros ECi , and glycated haemoglobin using DCA 2000 , Quality of life was measured by the heart specific MacNew questionnaire Allocation was done by a computer using block randomisation. The first, the smallest and the largest block, were defined by the technicians at the unit of Applied Clinical Research at the university. The person including the patients got the allocation results on screen and by e-mail by logging on to a website.2peak between the two groups. Secondary analyses were changes in VO2peak within groups, as well as changes in secondary outcome measures between and within groups. The mean change in each group from baseline to follow-up testing was reported as the estimated margin of the mean (EMM), with 95% confidence intervals (CI). Within-groups differences were considered significant when the 95% CI did not include zero The primary analysis of this study was changes in VO2peak of 28.1 mL·kg−1 min−1 and heart rate recovery of 20. At the baseline test we saw no medical reasons for this patient not to exercise according to the standard rehabilitation program or as home-based AIT. Apart from this event, we experienced no unfortunate side effects of the exercise training programs. Patients were recruited between October 2006 and November 2007, and follow-up tests were done between April 2007 and June 2008.Baseline characteristics of the patients are shown in The home exercise and residential group increased peak oxygen uptake with 18.8% and 17.4%, respectively , with noHealth related quality of life increased significantly within both groups, with non-significant between-group difference . We saw Medication at baseline is outlined in Patients in the AIT group registered their training throughout the follow-up period and did 1.6 (±1.6) AIT sessions and 2.4 (±1.9) moderate intensity sessions weekly. 2peak and quality of life after both residential and home-based AIT in patients undergoing coronary artery bypass surgery. We found no between-group differences in the increase in VO2peak and therefore no evidence for a different treatment effect between the two interventions. We have, however, no evidence of equal treatment effects of residential rehabilitation and home-based AIT based on our data. Also others 2peak in hospital versus home-based exercise groups after coronary artery bypass grafting, and a recent Cochrane review found home-based exercise to be as efficient as hospital-based programs The main finding of our study was significant improvements in VO2peak in the home-based group due to low adherence to exercise. We found however that some of the patients chose to do moderate exercise instead of AIT, and that some did both moderate exercise and AIT. We do not know of earlier studies investigating home-based AIT in cardiac patients without first attending a residential or policlinic rehabilitation program. However, we have in two previous studies 2peak during the follow-up period.According to training diaries, at least 7 (two incomplete registrations) of the 12 patients reported ≥2 weekly interval sessions the whole 6 month follow-up period. In general, there was an overall acceptable adherence to exercise in the home-based group, making us have to reject the hypothesis of inferior increase in VO2peak at baseline between the two groups, showing that their initial fitness level was similar. Only the residential group improved their levels of HDL cholesterol during the follow-up period, with a significant different change between groups. This was perhaps caused by the diet counselling and practical cooking sessions at the rehabilitation centre received by this group, in contrast to the written material on diet received by the home-based AIT group. The observed significant increase in glycated haemoglobin (HbA1c) in both groups despite participation in cardiac rehabilitation is difficult to explain. We saw no significant change in body weight during the follow-up period, so increased body mass was not the reason for this increase. One possible explanation, however, is that these patients are developing a reduced insulin sensitivity due to an on-going unhealthy lifestyle that also gave them coronary heart disease.The increase in heart rate recovery (HRR) observed only in the home-based AIT group, was probably caused by a significantly inferior HRR at baseline in this group. The already quite high HRR value at baseline in the residential group is a possible explanation of the lack of improvement in that group. A lower HRR at baseline may indicate that the AIT group was a sicker population than the residential group. However, there was no significant difference in VOOne limitation to our study was the lack of follow-up of elicited exercise intensity and volume after the stay at the rehabilitation centre in the residential group. Due to difficulty in enrolment of patients, our study was under-powered for the primary endpoint and this is also regarded as a limitation. Since our study was not adequately powered, it should serve to stimulate a larger multicentre randomised trial. Our study indicates however, that as little as one hour of instruction in how to do AIT leads to significant improvements in aerobic capacity at the follow-up test (after coronary artery bypass grafting). Furthermore, there is a further need for investigating how home-based exercise training can be arranged as a cost effective alternative to residential cardiac rehabilitation, and to establish the safety aspects of home-based AIT for cardiac populations.2peak in patients undergoing coronary artery bypass graft surgery. Further, our study suggests that home-based AIT is a feasible form of training in cardiac patients in a home setting. We have earlier found that patients randomised to AIT after myocardial infarction were able to maintain their VO2peak as long as 30 months after ending formal rehabilitation at the hospital, whereas patients following usual care declined significantly during this time We conclude that both residential rehabilitation and home-based AIT improve VOChecklist S1CONSORT Checklist.(DOCX)Click here for additional data file.Protocol S1Trial Protocol.(DOCM)Click here for additional data file.


VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease.Various missense mutations in the  VHL) disease, neurofibromatosis type 1 (NF 1), multiple endocrine neoplasia type 2 (MEN 2) and mutations in any one of the mitochondrial succinate dehydrogenase complex (SDHx) genes SDHAF2 and TMEM127 may also be associated with PCCs Pheochromocytomas (PCC) are rare tumors arising from the chromaffin-cells of the adrenal medulla. They might arise sporadically or in association with any one of the cancer syndromes that includes von Hippel-Lindau (VHL) disease, described first in the late 1800s, is an autosomal dominant disorder that could increase susceptibility to a range of tumors, both benign and malignant, including retinal hemangioblastomas, renal cysts and renal cell carcinoma, PCCs and other tumors VHL disease only in 1964 VHL disease VHL disease, it is a common feature associated with type 2 disease. Type 2 disease is further subgrouped into 2A, 2B, 2C where type 2C is known to be associated with only PCC. Also the pattern of mutations in these subgroups appears to be distinct VHL disease is known to be associated with deletions and truncating mutations while type 2 disease is associated with missense point mutations, which in turn is believed to reflect the degree of functional change it can cause to the VHL gene product VHL gene mutations were eventually detected when the testing system became available as part of a routine screening of patients with PCC. The presence of a novel mutation, never described before in germline configuration, among these patients, also prompted us to undertake a detailed in silico analysis of the mutation to determine its pathogenic/deleterious potential.von Hippel-Lindau , confirming the diagnosis of a right adrenal pheochromocytoma. He underwent right adrenalectomy and the post-operative period was uneventful. He was subsequently lost to follow. In 1979, he presented to us again with severe headache and recurrent seizures. On examination his pulse rate was 100/min, blood pressure was 160/130 mm of Hg. Cardiac auscultation revealed a loud 2He subsequently got married and had two children as shown in His eldest son presented to us at 10 years of age with severe hypertension. He was found to have bilateral pheochromocytoma. He underwent bilateral adrenalectomy in 1998. Subsequently he has remained asymptomatic.nd heart sound and a 3rd heart sound. A short systolic murmur was heard along the left sternal border. She had bilateral papilledema with hemorrhages & exudates, and a macular star. There was no corneal nerve thickening or retinal angiomas. Rest of the systemic examination was normal. 24 hours urinary VMA on 2 occasions was elevated 8 and 8.1 mg . MIBG scan . Twenty picomoles of primers were used for amplification of the VHL exons 1, 2 and 3 using previously published primers www.sangers.ac.in and www.lovd.com databases were checked to verify if the mutation . The study commenced in 2010 when genetic analysis for mutation  was prevIn silico analysis is a widely used approach for the prediction of unclassified variants as pathogenic or benign based on prediction scores. Most of the in silico tools utilize sequence, structure information along with physical properties of amino acids to calculate the prior probability of pathogenicity for unclassified variants. Six widely used mutation pathogenicity prediction tools were used for analysis in this study. Evolutionary information based SIFT + or Cl– ions. The temperature was kept constant by using a Berendsen algorithm The potential impact of the mutation p.Arg82Leu was evaluated by visual inspection of environment of the altered residue in the crystal structure of the VHL protein. The structure of the VHL protein (PDB ID 1LM8) contains two primary functional domains: the α-domain (155–192 aa), and the β-domain (63–154 and 193–204 aa) Structural properties of the native and mutant model of VHL protein were calculated from the trajectory files with the built-in functions of GROMACS 4.5.3. The trajectory files were analyzed through the use of g_rmsd and g_rmsf GROMACS utilities in order to obtain the root-mean-square deviation (RMSD) and root-mean square fluctuation (RMSF) values. Number of distinct hydrogen bonds formed in protein during the simulation was calculated using g_hbond utility. Number of hydrogen bond determined on the basis of donor-acceptor distance smaller than 3.9 nm and of donor–hydrogen-acceptor angle larger than 90 nm et alThis is the first report of a germline p.Arg82Leu (c.245 G>T) missense mutation being reported among three patients from a family with bilateral pheochromocytoma . This murd or 4th decade of life.3 However, the mean age of diagnosis of PCC in VHL disease is much lower at ∼20 years with a clear predisposition to an early onset of tumors VHL gene Patients with PCC normally present during their 3VHL gene, identified in 1993, is located on chromosome 3p25–26. It is a tumor suppressor gene that encodes a protein that regulates hypoxia-induced proteins VHL gene in all cells. For VHL-associated tumors to develop there must be loss of expression of the second normal allele (second hit), through either somatic mutation or deletion of the second allele, or through hypermethylation of its promoter. More than 500 germ line mutations have been identified to date VHL gene product is believed to differ with different VHL mutations and mutations that do not cause a total loss of function but are associated with a risk of pheochromocytoma, have been described previously in silico approach, to determine its potential in being deleterious/pathogenic. Different working groups have applied various in silico methods as an integrated model to assess the pathogenicity of unclassified variants The in silico tools namely SIFT, I-Mutant3, PhD-SNP, MutPred, PolyPhen2 and SNPs&GO in order to determine the pathogenic potential of the variant. Identifying variations and determining the pathogenic potential of single nucleotide polymorphisms (SNPs) responsible for specific phenotypes is a difficult task in the laboratory. However, the availability of sophisticated computational tools that efficiently discriminate the pathogenic variants from benign in a fast and accurate manner has changed the scene phenomenally. Here, we analyzed VHL variant p.Arg82Leu with six different Protein three dimensional (3D) structural information is an important feature for predicting the effects of deleterious variant and also provides information about the environment of the mutation. Proteins with mutations do not always have 3D structures that are analyzed and deposited in Protein data bank (PDB) necessitating the construction of 3D models by locating the mutation in 3D structures. This is a simple way of detecting the adverse effects of a mutation on a protein. Several researchers have utilized the structural information along with molecular dynamics to predict the impact of mutations in proteins and protein complexes To evaluate the structural flexibility of both native and mutant structures of VHL protein, we calculated the Root Mean Square Fluctuations (RMSFs) values from the 12 ns simulation trajectory data. The RMSFs values of native and mutant structures are shown in Interaction between positive and negative ions in proteins lead to salt bridges, which is an important stabilizing force and the presence of salt bridges, is an evidence for close proximity in the structure. Salt bridge distances of VHL protein in both native and mutant states were calculated from the 12 ns trajectory data and shown in 2 to ∼57 nm2 in the whole 12 ns simulation period. But all the mutant structure p.Arg82Leu obtained less SASA of ∼44 nm2 to ∼54 nm2. In comparison with the native protein, the mutant proteins obtained less SASA. Reduced SASA in the mutant proteins indicate that there may be change in the accessible area of amino acid residues from exposed to buried state vice versa. Lesser accessible area decreases the probability of regular interaction with biomolecules. Summing, all the in silico findings using different parameters such as RMSD, RMSF, salt bridge, H-bonds and SASA applied in molecular dynamic study, the variant p.Arg82Leu was found to alter the structure of the protein and to have a pathogenic potential.The solvent accessible surface area (SASA) of a biomolecule is that surface area traceable by the solvent molecule. These solvation effects can be calculated by explicit solvent models, such as MD simulations using a sphere of water molecules All the three patients with the p.Arg82Leu mutation have been on long follow-up, with the index case being followed up for ∼40 years. Interestingly, all these patients have remained asymptomatic despite the prediction tools used in this study predicting the mutation to have a pathogenic potential. Further, analyzing the conformational changes of amino acid residue within VHL protein has helped to identify significant structural changes. Whether these patients will eventually develop symptoms or remain asymptomatic through their lifetime, indicating that the risks associated with carrying the p.Arg82Leu mutation is minimal, is a feature that can only be figured with a passage of time.


As schizophrenia is genetically and phenotypically heterogeneous, targeting genetically informative phenotypes may help identify greater linkage signals. The aim of the study is to evaluate the genetic linkage evidence for schizophrenia in subsets of families with earlier age at onset or greater neurocognitive deficits.Patients with schizophrenia  and their first-degree relatives  from 557 families with schizophrenia were recruited from six data collection field research centers throughout Taiwan. Subjects completed a face-to-face semi-structured interview, the Continuous Performance Test (CPT), the Wisconsin Card Sorting Test, and were genotyped with 386 microsatellite markers across the genome.A maximum nonparametric logarithm of odds (LOD) score of 4.17 at 2q22.1 was found in 295 families ranked by increasing age at onset, which had significant increases in the maximum LOD score compared with those obtained in initial linkage analyses using all available families. Based on this subset, a further subsetting by false alarm rate on the undegraded and degraded CPT obtained further increase in the nested subset-based LOD on 2q22.1, with a score of 7.36 in 228 families and 7.71 in 243 families, respectively.We found possible evidence of linkage on chromosome 2q22.1 in families of schizophrenia patients with more CPT false alarm rates nested within the families with younger age at onset. These results highlight the importance of incorporating genetically informative phenotypes in unraveling the complex genetics of schizophrenia. Family and twin studies have indicated that schizophrenia has a strong genetic basis Cumulative evidence suggests that age at onset is one potential component phenotype for schizophrenia. Compared with schizophrenia patients with late onset, those with early age at onset have been associated with more severe cognitive impairments and poorer outcomes A number of candidate gene studies have explored the relations of certain genetic variants to schizophrenia by subgrouping patients using these genetically informative phenotypes of schizophrenia, including age at onset This study aimed to evaluate the genetic linkage evidence for empirically derived subtypes of schizophrenia by applying ordered subset linkage analyses in a large sample of families of siblings co-affected with schizophrenia, which consisted mainly of a single ethnic group Participants of this study included patients with schizophrenia and their first-degree relatives recruited from six data collection research center throughout the nation in the Taiwan Schizophrenia Linkage Study between 1998 and 2002. The study design, ascertainment process, and sample characteristics have been described in more detail elsewhere The DIGS includes a psychosis section that inquires about the age at onset of the first psychotic episode that could not be attributed to medical illness, medications, or substance abuse. When the information with regard to onset of the first psychotic episode on the interview was unclear, research psychiatrists would review the medical history and determine the age at onset.Probands and their relatives completed both the CPT and the WCST. The degraded and undegraded version of CPT -‘1-9’ procedure has been described in detail elsewhere For the WCST, we used a computerized version The adjusted z scores of the CPT indices were derived by means of standardizing the raw scores with adjustments for sex, age, and education against a community sample of 345 individuals http://www.cidr.jhmi.edu). A series of procedures for quality control was adopted for the genotyping, such as duplicate assays with a discordancy rate of 0.06%, and checking for non-Mendelian inheritance and pedigree inconsistencies with an overall rate of 0.39% in genotype and family errors http://zork.wustl.edu/nimh.The original genotyping was conducted by the Center for Inherited Disease Research, with 386 markers spaced at an average of 9-cM intervals, following the center's standard genotyping procedures , and three WCST indices . Meanwhile, the remaining indices were ranked using the descending order: four CPT indices  and five WCST indices .P value of ≤ 0.05 was considered significant, suggesting that a particular subset of individuals in the sample is more strongly linked to the specified chromosomal region than the whole sample. The procedures are summarized in the left part of The statistical significance of the difference between the greatest LOD scores in a subset of families identified by the OSA and the LOD scores at the same position in the full set of families was evaluated via 1,000 permutations. Under the null hypothesis that the ranking of the covariate is independent of the family's LOD scores on the target chromosome, the families were randomly permuted with respect to the covariate ranking and a chromosome-wide p value for each chromosome was yielded. A P value of <0.05 in permutation was considered significant.Because OSA-derived subsets of families might have tied covariates, we attempted to introduce another subphenotype to further dissect the families , and thus the threshold of a corrected genome-wide significance level was set as 0.0029 . For the nested OSA part, there were 33 covariates examined (17 covariates in the OSA and 16 indices on the CPT and the WCST), and the threshold of a corrected genome-wide significance level was set as 0.0015 .After the permutation on the composition of a subset of families, which provides chromosome-wide significance level, we further evaluated its genome-wide significance by simulating the genotype data of the families and then estimating the empirical significance level with correction for multiple phenotypes, as illustrated in the right part of ad hoc correction procedure was also performed, which was calculated as LOD(corrected)  =  3.6 + log10 (numbers of tests) Second, according to the Lander and Kruglyak threshold The family covariate values between the subset of families that resulted in the greatest linkage signals and the remaining families were then compared. A linear mixed effect model with family treated as a random effect was applied using the Proc MIXED of the SAS software version 9.1  to adjust for within-family correlation.P<0.0001), 0.14 to 0.31 for CPT indices (P ranging from 0.004 to 0.0001), and 0.12 to 0.24 for WCST indices (P ranging from 0.04 to 0.0001).The distributions of individual covariates, which were averaged over the affected siblings of each family, are listed in P ≤ 0.05) in a subset-based maximum LOD score from the initial LOD scores of the whole sample. The results of the OSA identified distinct chromosomal regions implicated for schizophrenia with different informative phenotypes: 1) 2q22.1 for younger age at onset; 2) 7q32.2, 8q13.1, and 9q34 for greater undegraded CPT-related deficits; 3) 7q32.3 for greater degraded CPT-related deficits; and 4) 8q13.1 and 8q21.1 for greater WCST-related deficits. For the five loci with significant increases in subset-based LOD scores, the percentage of linked families in each subset ranged from 10% to 53% of the whole sample. When the families within the subset were compared with the remaining families for the corresponding subphenotype, there were indeed significant differences between the two groups in the covariates, with earlier age at onset or more neurocognitive deficits in the subset of families. Of those covariate/locus combinations with a significantly increased LOD score, only “age at onset/2q22.1” reached genome-wide significance based on simulations after an ad hoc correction for multiple informative phenotypes, whereas no locus reached the Lander and Kruglyak genome-wide significance threshold after an ad hoc correction for multiple informative phenotypes.P = 0.004) in 81% of 282 families  and 7.71  in 87% of 281 families , respectively. Of note, both the LOD score of 7.36  and of 7.71  reached genome-wide significance, regardless of which method was used in the significance evaluation, i.e., simulated genotype data or the adjusted Lander and Kruglyak threshold after accounting for multiple phenotypes. More severe CPT or WCST deficits were indeed found in the families of the nested subset than those in the remaining families, as summarized in Among the loci identified in The distributions of the genome-wide LOD scores of the nested OSAs that were based on age at onset and CPT false alarm rates over 22 autosomal chromosomes are illustrated in This study found that subsets of families of patients with schizophrenia characterized by younger age at onset or neurocognitive deficits exhibited increased linkage signals and reached genome-wide significance on 2q22.1, which was nearby a suggestive locus (a LOD score of 1.16 on 2q14.1) in the initial genome-wide scan for the whole sample Our nested OSA approach demonstrates an exploratory way to incorporate various schizophrenia-related characteristics in deciphering the genetic linkage signal to schizophrenia for families with tied covariates . Our results revealed that in the onset-based subset of families having maximal linkage signals, families with tied covariates might in fact have linkage scores in the opposite directions. Accordingly, a further subsetting by one CPT or WCST index might partition the families into more homogeneous subsets and then result in more increase in the LOD scores. Unlike other statistical methods for detecting linkage by dissecting heterogeneity, which require a prior stratification of the data An important feature of this study is that we used age at onset and several facets of the CPT and WCST as covariates to reduce phenotypic and genetic heterogeneity of schizophrenia. These are important features as they clearly impact the outcome and severity of the illness. In addition, cognitive deficit is a core symptom for schizophrenia Unlike previous linkage studies for psychiatric disorders that used selective indices of neurocognitive tasks, either as a composite profile of neurocognitive performance P values were derived from simulations. The current results of particular subset-based NPL-Z scores attaining genome-wide significance might be very conservative because a Bonferroni-type correction of the P value was applied for the correlated indices among the CPT or WCST. It should be pointed out that it is very difficult to assess the statistical significance of our subset-based linkage signals due to the complexity inherent in the many possible sources of multiple testing. Accordingly, the results reported here might be better treated as exploratory and independent replication is warranted to reassure the robustness of the findings.However, our approach might suffer from the issue of multiple testing. To fully address this, several strategies were used in this study to help interpret the significance of the OSA results. First, we used permutation tests to estimate the statistical significance of the change in LOD score from the subsets, which controlled for inflation in the false-positive rate induced by examining multiple family subsets for a given covariate. Second, two simulation-based strategies were applied to estimate the genome-wide significance level of subset-based LOD scores, with one based on the simulated genotype data of this sample and the other one based on adjusting the Lander-Kruglyak threshold that was also obtained by simulations. Such simulations can help control for the inflation in the false-positive rate over multiple chromosomes and markers since genome-wide significance was estimated by the proportion of times that a particular LOD score was reached anywhere in the simulated genome. Finally, we used a Bonferroni correction to adjust for multiple trait-related covariates after empirical genome-wide The most striking linkage signal found in this study was obtained from a nested OSA on chromosome 2q22.1 (D2S4422 at 147.4 cM). Intriguingly, a rank-based meta-analysis showed evidence of linkage in this region This study is limited in several aspects. First, our samples consisted of adults with Han Chinese ancestry only, and hence the results may not extrapolate to populations of other ethnicities where allele frequencies may vary. Second, despite our use of different strategies to deal with multiple testing, our linkage findings may not rule out the possibilities of false positive or false negative because genome-wide statistical significance of OSA-derived subset results remains a difficult issue. Third, the age at onset of schizophrenia in this study was determined mainly on the initiation of first psychotic symptoms. Although the age at first functional impairment may be closer to the true onset of the disease, there is an overall agreement that the onset of the first psychotic episode is a better marker for the onset of schizophrenia because it has a higher specificity than negative or nonspecific symptoms In summary, we obtained possible evidence of linkage on chromosome 2q22.1 in families of schizophrenia patients with more CPT false alarm rates nested within the families with younger age at onset. Our results suggest the presence of etiologic heterogeneity in schizophrenia and highlight the importance of unraveling the complex genetics of schizophrenia by incorporating genetically informative phenotypes.


This study examined the psychometric properties of the Cardiac Depression Scale (CDS) in a sample of coronary heart disease (CHD) patients.A total of 152 patients were diagnosed with coronary heart disease and were administered the CDS along with the Beck Depression Inventory- 2 (BDI-2) and the State Trait Anxiety Inventory (STAI) 3.5-months after cardiac hospitalization.r = 0.64). More patients were classified as severely depressed using the CDS. Both the CDS and the BDI-2 displayed significantly strong correlations with the STAI (r = 0.61 and r = 0.64), respectively.The CDS’s factorial composition in the current sample was similar to that observed in the original scale. Varimax-rotated principal-components analyses extracted six factors, corresponding to mood, anhedonia, cognition, fear, sleep and suicide. Reliability analyses yielded internal consistency α - coefficients for the six subscales ranging from 0.62 to 0.82. The CDS showed strong concurrent validity with the BDI-II (These findings encourage the use of the CDS for measuring the range of depressive symptoms in those with CHD 3.5 months after cardiac hospitalization. A high incidence of depression has been reported in those with coronary heart disease (CHD) specifically in those who have had coronary artery bypass graft surgery-3, thoseA range of depression assessment tools have been used to measure depressive symptoms in CHD patients. These include the Beck Depression Inventory – 2 (BDI-2), the HosThe CDS has been developed as a depression scale to accurately assess depression in cardiac patients. The oriThe CDS has been employed to assess for a range of depressive symptoms in cardiac patients with various diagnoses and at different time points including use with ambulatory cardiac outpatients with a range of diagnoses including angina, heart failure, post-myocardial infarction, valvular heart cardiac disease and arrhythmias; those wHowever, further validation of the CDS in a CHD population who are medically stable and have settled into their community surroundings after cardiac hospitalization is needed given that studies have shown that around one third of those with CHD meet criteria for depression 3–4 months after a cardiac event. The curM = 70.34 years, SD = 8.18). TableOne hundred and fifty two outpatients with CHD who had been admitted for a cardiac procedure were recruited from two Coronary Care Units in Melbourne, Victoria. Recruitment took part between June, 2009 and September, 2011. Initial recruitment took place while the patient was still in hospital or straight after discharge. Of the 356 patients admitted to the coronary care units of two hospitals which were approached, 105 patients were not contactable and 99 did not want to participate, resulting in a final sample of 152 patients (response rate: 59%). The 152 patients who participated in this study consisted of 101 males (66.4%) and 51 females (33.6%) and ranged in age from 45 to 92 years  to ‘strongly agree’ (7). Participants are asked to rate how strongly they agree or disagree with each statement. A scale point of 4 indicates neither disagreement nor agreement with the item. Seven items are reverse-scored. Higher scores on the CDS indicate more severe depression. The CDS has fewer items that refer to somatic symptoms of depression compared to the BDI-2. The total CDS score is the sum of all items and ranges from 26 to 182. Cut-off scores to indicate mild, moderate or severe depression were not provided by the original authors for the CDS. However, a cut-off score of 90 for mild depression and 100 or above has been recommended to detect individuals with more severe depression[the past two weeks, including today’. Scores range from 0 to 3 with 0 indicating absence of the symptoms and higher values indicating greater severity of the symptom. The BDI-2 score can be grouped into mild (10–19), moderate (20–25) and severe (>25) symptoms[The 26-item Cardiac Depression Scale (CDS) is desigpression, Wise etpression is a widsymptoms. The intin the past two weeks, including today’. Responses are on a 4-point Likert scale ranging from (0) ‘not at all’ and (3) ‘very much so’ with higher scores reflecting higher severity. In the current sample, the STAI showed excellent reliability (α = 0.93).The State-Trait Anxiety Inventory (state version) (STAI) is a 20-The 26-item World Health Organisation Quality of Life scale (Brief version) (WHOQOL-BREF) was usedThe 12-item Perceived Social Support Scale (PSSS) assessesIn order to reproduce the factor structure reported previously we undertook the factor analysis procedures used by Hare and Davis (1996) and WiseThe total 26-item CDS scale showed good reliability (α = 0.91). As seen in Tabler > = 0.3). A significant moderate to high correlation between the CDS and the BDI-2 scale  and between the BDI-2 and the STAI  was observed. Similarly, moderate to high correlations were found between the CDS and the STAI scale , the CDS and the physical QOL subdomain , psychological QOL subdomain , social QOL subdomain  and environmental QOL subdomain , and the PSSS . A weaker correlation was found between the CDS and the ESSI, .Pearson’s correlation coefficients were used to established the CDS’s concurrent validity with a moderate to high correlation between the relevant scales deemed as acceptable [There is growing evidence that the CDS is characterized by adequate psychometric properties when applied to cardiac samples. Reliability has been found to be high across cardiac samples when assessed in terms of internal consistency . Moreover, the validity of the CDS has been used successfully to discriminate between cardiac patients diagnosed as depressed vs. nondepressed. The CDS has also been found to have good concurrent validity with the BDI-2 (ranging from 0.69 to 0.73) and the STAI ( = 0.80),26,27. C = 0.80),24,27. T = 0.80). The mod = 0.80). The intIt should be noted that the current sample of CHD patients reported a high CDS mean score compared to previous cardiac samples. This maIn the current study, depressed participants were determined by a BDI-2 score of 10 or more. Both the CDS and the BDI-2 identified that almost one in four patients (25% vs. 24.3%) had symptoms indicative of mild to moderate depression. These results are comparable to those reported by Wise, Harris and Carter (2006) who founWhile the BDI-2 suggested that 14.5% had symptoms indicative of severe depression, the CDS indicated that in the same cohort, 27.6% of patients were suffering severe depression which is almost one in three patients as opposed to almost one in six by the BDI-2. This discrepancy may be due to the CDS items detecting more severe symptomatology compared to the BDI-2, the cut-off score used for detecting severe depression with the CDS and the possibility that the CDS may be overdiagnosing severe depression in this sample. However, these results are comparable to previous research which found that the CDS classified 21-25% of cardiac outpatients as severely depressed,15,18,27A major limitation of the current study is that no diagnostic interviews to assess for Major Depressive Disorder or Episode were concurrently undertaken with participants to validate the levels of depression found with the CDS and the BDI-II. The current study was not able to evaluate the sensitivity or specificity of the CDS in a fully meaningful way. Further research employing larger sample sizes is required to determine whether the CDS is a more sensitive measure of depression in the 3–4 month time period after a cardiac event or whether the CDS is yielding more false positives. The current results need to be replicated given that the current sample was less than what has been recommended for conducting factor analysis with suggestions that a minimum sample size of 200 be used. Future Another limitation is that no information was gathered from those that declined to participate and no comparison can be made between this sample and those that agreed to participate in the current study.Effective screening after a cardiac event and procedure has implications for the effective management of depression to assist cardiac patients in their therapeutic compliance and in their prognosis. Given that those in the current sample who were depressed also scored lower on the social support and quality of life measures, overall treatment approaches should focus on strategies to increase social supports and quality of life in these patients.Depression has been shown to influence health outcomes in those with CHD. Accurate and timely assessment of depression in CHD patients has the potential to influence prognosis and reduce suffering in those who have been discharged after cardiac hospitalization. The current study has shown that the CDS can be used to screen and detect the range of depressive symptomatology, ranging from mild to severe, in CHD patients who are medically stable and settled in their community surroundings 3.5 months following discharge after cardiac hospitalization.The authors declare that they have no competing interests.LK contributed to the conceptualisation and design of the research, the data collection, analysis and interpretation of the study results and in the writing of the manuscript. IM, AT and DC contributed to the design of the study, contributed their clinical and research expertise in the areas of assessment, depression and CHD and provided assistance in the interpretation, translation and write up of the findings. PA and JP contributed to design and data collection. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/12/216/prepub


Postal questionnaires are simple and economical for collecting outcome data for randomised controlled trials (RCTs) but are prone to non-response. In the RECORD trial (a large pragmatic publicly funded RCT in UK) non-responders were sent a reminder and another questionnaire at 1 year, of which 40% were returned. In subsequent years we investigated the effect of an advance telephone call to non-responders on responses rate to reminder questionnaires and the next questionnaire 4 months later.n = 390) or to a control group that received the usual reminder schedule only (n = 363). The primary outcome was response to the reminder questionnaire within 21 days; secondary outcomes were response to a questionnaire 4 months later; completeness of quality of life instruments; and the number of participants declining further follow-up. Results are presented as odds ratios from a logistic regression intention-to-treat (ITT) analysis and then percentage difference and 95% confidence intervals (CI) for both ITT and average treatment effect on the treated (ATT) analyses.Non-responders to annual questionnaires were randomised to receive a telephone call from the trial office ahead of the reminder questionnaire in addition to the usual reminder schedule (The proportions that responded were 67.8% (265/390) in the intervention group compared to 62.5% (227/363) in the control group. The ITT estimate was a 5.4% increase (95% CI −1.4 to 12.2). Four months later percentages responding were 51.8% (202) and 42.7% (155). The ITT estimate was a 9.1% increase (95% CI 2.0 to 16.2). In the intervention group 12.3% (48/390) of participants were not telephoned because questionnaires were returned before the scheduled telephone call. ATT estimates adjusting for this were 6.2% (95% CI −1.6 to 14.0) and 10.4% (95% CI 2.2 to 18.5), respectively.The telephone call resulted in a slight increase in response to the reminder questionnaire, however at 4 months later the proportion in the telephoned group responding was greater. This study suggests that pre-notification telephone calls may only be worthwhile if further questionnaires are to be sent out soon after reminder questionnaires.ISRCTN51647438Current Clinical Trials  Postal questionnaires are simple and economical for collecting outcome data for randomised controlled trials (RCTs) but achieving high response rates can be very challenging. The pitfalls of poor response rates are well documented and include loss of outcome data, reducing the effective sample size (and hence precision) of the trial and increasing the potential for bias .Several strategies have been used to try to maximise response rates to research studies, including pre-notification and the use of incentives, and these have been widely reported. For example, the Cochrane Review by Edwards and colleagues looking at methods to increase response rates to postal and electronic questionnaires  identifiThe RECORD trial  involved people aged 70 years or over who had a previous fracture. RECORD participants were sent questionnaires every 4 months that recorded data on self-reported fracture and quality of life . Annual We were already telephoning participants that did not respond within 3 weeks of their reminder questionnaire, which was well-received by these participants and resulted in an extra 41 responses. It was unclear whether the addition of a telephone call to participants prior to sending the questionnaire  would increase this further. We therefore embedded a methodological trial, the Phone Trial, of pre-notification telephone calls by randomly allocating non-responding participants to receive a pre-notification telephone call, or to receive RECORD’s usual reminder schedule.Participants in the RECORD trial who did not respond to the initial mailing of the annual questionnaire at 12- and 24-month follow-up, and were not randomised in other ongoing methodological sub-studies were considered eligible for the Phone Trial. Eligible participants were stratified by their RECORD trial allocation and randomised using a computerised central allocation process at the Trial Office to one of the following:–The intervention group: Telephone call from the Trial Office up to 14 days before the questionnaire was sent, asking the participant to complete all the questions on the questionnaire when it arrived. This was followed by the usual RECORD reminder schedule (see below).Or–The control group : Repeat mailing of usual follow-up letter and the questionnaire, followed by a phone call by the study nurse or the Trial Office if the questionnaire was not returned within 3 weeks.The primary outcome of the Phone Trial was the proportion of reminder questionnaires returned within 3 weeks of the reminder being sent. Secondary outcomes were proportion of questionnaires returned at the next time-point 4 months later ; completeness of data for the EQ-5D and SF-12 quality of life measures (QoL) in annual questionnaires; and the number of participants declining further follow-up. We were interested in measuring the completeness of the QoL measures as these were secondary outcomes collected from everyone (the primary outcome was a rare outcome whose completeness could not be assessed) in the RECORD trial, in case people were more likely to return incomplete questionnaires. We also thought that the phone call might offer participants an extra opportunity to decline further follow-up so this potentially negative impact was also of interest.The sample size available was constrained by the number of eligible participants in the RECORD trial and a formal sample size calculation was not used to derive a target sample size for the Phone Trial.If a participant was randomised more than once  only the first year response data were used. Descriptive data on the baseline characteristics are summarised using means and standard deviations (SD) and counts and percentages, where appropriate. All outcomes were analysed using logistic regression and effect sizes are presented as both ORs and absolute percentage differences (with 95% CIs) to aid interpretation. Analyses were by ITT, that is all participants were included regardless of whether they received the planned phone call or not. In the intervention group there were participants who did not receive the phone call  compared to 227/363 (62.5%) in the control group. The ITT estimate of the between-group difference was a 5.4% increase (95% CI −1.4% to 12.2%) in the intervention group. The ATT estimate, which accounted for the 48 participants in the intervention group who did not need to receive the phone call, was 6.2%   and 1.8%  for EQ-5D and SF-12, respectively, with both results being higher in the intervention group. There were nine intervention group participants who withdrew on the day they received their phone call. However, there was no evidence of an increase in the number of participants declining further follow-up  but the uncertainty around this estimate does not rule out the possibility that the intervention may actually harm response rates.There are a number of possible reasons for this. First, the control group response rate at 62.5% was higher than anticipated. When the Phone Trial was first conceived only 40% of participants were responding to reminders. There was therefore a potential ceiling effect, with the maximum achievable response rate having already been reached, or almost reached, because of other response rate initiatives that were ongoing prior to the Phone Trial. Second, the 3-week cutoff for response may have been too short. This was chosen because a further reminder strategy  was implemented within the RECORD trial for persistent non-responders after this time period had elapsed. Third, there was no pre-specified power calculation. Methodological studies to improve response rates are constrained by size of the parent trial in which they are embedded. If we consider the observed effect in the Phone Trial, a small but potentially worthwhile increase in response of 5.4%, then the sample size required  would be approximately 4,000 participants. Sample sizes of this order are hard to achieve for embedded methodological trials so it is essential that they are published, with clear methods and intervention descriptions, so that meta-analysis is possible.a priori concerns about the Phone Trial intervention. First, there was a concern that although responses may increase this would come at the expense of partial completion of QoL instruments, leading to unusable data. This is a possibility; for example, the proportion of the 265 responding to the reminder that had usable EQ5D data was 86% (228/265) in the Phone Call group, this proportion was 88% (200/227) in the control group. Second, there was a concern that the participants might use the pre-notification phone call as an opportunity to decline further follow-up. There were only nine individuals in the intervention group that did this; for the next 12 months the groups had very similar patterns of declining further follow-up. Third, we anticipated that due to the scheduling logistics of making the phone calls that some participants may not actually receive phone calls. We accounted for this in our analysis by using an ATT estimate in addition to the standard ITT estimate. In fact the intervention was well implemented and the results of the ITT and ATT analyses are similar.We had three The review by Edwards et al.  estimateThe telephone call resulted in a slight increase in response to the reminder questionnaire, however at 4 months later the proportion in the telephoned group responding was greater. This study suggests that pre-notification telephone calls may only be worthwhile if further questionnaires are to be sent out soon after reminder questionnaires.The authors declare that they have no competing interests.GMac analysed and interpreted the data, and drafted and revised the manuscript. AM conceived the idea for the study, managed data collection and delivery of the intervention, interpreted the data, and drafted and revised the manuscript. GMc conceived the idea for the study, managed data collection, analysed and interpreted the data, and drafted and revised the manuscript. ST interpreted the data, and drafted and revised the manuscript. AA conceived the idea for the study, managed data collection and delivery of the intervention, interpreted the data, and drafted and revised the manuscript. All authors read and approved the final manuscript.


Glued), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11).Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150To reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons.The dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events. Dynactin is a multisubunit protein complex in eukaryotic cells required as an activator of cytoplasmic dynein, the major minus end-directed microtubule motor,2. DynacDynactin is composed of eleven different subunits ranging in size from 22 to 150 kDa. SeveralGlued), four dynactin2 (p50 or dynamitin), and two dynactin3 subunits . The Arp1 rod is built of eight Arp1 molecules forming a short actin-like filament, probably one β-actin molecule, and the conventional actin capping proteins Capα and Capβ, which are located at the barbed-end of the mini-filament. The other end of the filament is terminated by Arp10  and dynin vivo causes disruption of the dynactin complex[Dynactin1 is the largest subunit of the dynactin complex and belo complex,28,30. D complex. Arp1 is complex that rep complex,32,33.Neurospora crassa[Saccharomyces cerevisiae[Drosophila melanogaster[Caenorhabditis elegans[Glued) gene in Drosophila species[The first studies on dynactin have been performed with chicken brain samples,2. Subsera crassa-39, Saccerevisiae-43, Drosanogaster,45 and Cs elegans. Althoug species.http://www.cymobase.org).Building such a multi-protein complex with a filament of fixed size seems rather complicated. Because most of the analyses of the complex have been done with vertebrate samples, it would be interesting to see whether the various unicellular protists that often have smaller gene repertoires, may have evolved compacted versions of the dynactin complex. Vice-versa, there could have been a minimal dynactin complex at the origin of the eukaryotes that multicellular eukaryotes expanded to accomplish more and different tasks. Here, we examined every known protein of the complex and determined its absence and presence in all eukaryotic genomes as available in September 2011. Furthermore, we inspected all genes to identify alternatively spliced exons and their appearance during evolution. For our analysis, we manually assembled and annotated more than 4,700 dynactin and actin-related protein sequences from about 550 species. All sequences were inspected and validated at the genomic DNA level to remove wrongly predicted sequence regions, to manually fill gaps in gene predictions, and to reveal the correct exon/intron boundaries. The sequences and related data like gene structure reconstructions and biochemical properties are available through CyMoBase  were termed “Partials”. “Partials” are not expected to considerably influence the phylogenetic tree computations. Sequences of which more than 5% were missing due to genome assembly gaps or incomplete EST data but that are otherwise unambiguous orthologs or paralogs were termed “Fragments”. "Fragments" are important to denote the presence of the subunits in the respective species in the qualitative analysis. Dynactin genes were termed pseudogenes if they contain more features like frame shifts and in-frame stop codons and miss more conserved sequence regions than can be attributed to sequencing or assembly errors.Petromyzon marinus to reveal at which stage alternative splice forms had been evolved in vertebrate evolution. To infer the phylogenetic relationship of duplicated dynactin subunits we calculated phylogenetic trees using the Maximum Likelihood and Bayesian methods. Gene structures were reconstructed for all sequences using WebScipio[http://www.cymobase.org) for further investigation.In total, the dynactin dataset contains 3061 sequences from 478 organisms Table , of whicebScipio and can Naegleria gruberi, and the Apusozoa Thecamonas trahens  the dynactin1 gene has been duplicated once[Brachydanio rerio, Takifugu rubripes, and Gasterosteus aculeatus. Some of the nematods like Brugia malayi also encode two versions of dynactin1. Two duplications of dynactin1 were found in the genome of the fungus Rhizopus arrhizus, and one additional dynactin1 in Mucor circinelloides. The variant A and B subunits each grouped together, suggesting a gene duplication predating the separation of the two species. Variant C of Rhizopus arrhizus grouped to variant B indicating another Rhizopus-specific duplication.Duplicates of dynactin1 have been found in independent branches of the eukaryotic tree exon 5 included, resulting in four additional positively charged residues exon 7 included, three additional positively charged residues exon 5 and 7 included, seven additional positively charged residues (Δ6)exon 6 and 7 included, five additional positively charged residues (Δ5)exon 5, 6 und 7 included, nine additional positively charged residuesWe did not find EST or cDNA-data for transcripts including only exon 6 , or EST-data including exons 5 and 6 without exon 7 (Δ7). Exon 27 is also a differentially included exon. Maybe because of lack of more full-length cDNA data or maybe because of tight regulation, exon 27 is found to be absent in dynactin1-Δ5,6,7, and to be present in dynactin1-Δ5 and dynactin1 . Thus, this proposed second EB1-binding site could be specific to mammals or, most likely, be an artefact from crystal packing effects. The latter is supported by another crystal structure of the complex of the dynactin CAP-Gly domain and the C-terminus of EB1, in which only the C-terminal EEY motif binds to dynactin1[The sequence conservation plot across all dynactin1 CAP-Gly domains shows that the core structure consisting of six beta-strands and several key residues for binding microtubule plus end-tracking proteins is highly conserved Figure D. The keynactin1. Severalynactin1) and theynactin1. All mutynactin1. The twoOgataea parapolymorpha and Ogataea angusta. Because two different species of Ogataea have been sequenced it is unlikely that dynactin2 could be missed because of gaps in the assemblies. None of the genomes analysed encodes more than one functional dynactin2 gene. Some mammals and Caenorhabditis brenneri contain dynactin2 pseudogenes.Dynactin2 was found in almost all branches of the eukaryotic tree that contain a dynactin complex Table . The onlHomo sapiens is encoded in 16 exons on chromosome 12 . AlthouThe residues encoded by the alternative exons (residues 36 to 40) are located in the N-terminal region of dynactin2 but have not been the specific focus of any biochemical study yet. Both the N-terminal and the C-terminal part of dynactin2 are needed for proper self-assembly and binding to dynactin3. The N-terminal 100 residues seem to be required and sufficient for binding to Arp1,67. BindNaegleria gruberi, Bigelowiella natans, Ciliophora, plants, and Stramenopiles  or because the scores of short exon hits were too low to be detected with TBLASTN.We were not able to identify dynactin3 homologs in Ustilaginomycetes, Chytridiomycota, s Table . Dynacti Figure . BecauseRattus norvegicus. The translations of both genes are identical except for three amino acids that are conserved substitutions. However, the gene of homolog B does not contain any introns and is not supported by EST data. Therefore, it is most likely the result of a recent retro-transcription of a processed pseudogene. Human dynactin3 is encoded on chromosome 9 in 7 exons, which are constitutively spliced  contains a sequencing error that led to a predicted N-terminal extension of 173 residues[Neurospora species and the closely related Sordaria macrospora but not in other Sordariales  or any other fungi. The sequence starting from the second methionine is highly conserved in all fungi and thus this methionine is most probably also the translation start site in Neurospora and Sordaria , tunicates , and other invertebrates. The exon invention event therefore predates the separation of the Gnathostomata and the Hyperoartia. There is not enough EST/cDNA data available to proof the alternative character of the exon in all vertebrates. For example, there is only one EST clone from Petromyzon that covers the respective sequence region and includes exon 6 but none without exon 6. But because there are EST/cDNA clones for several fish, mammals, and Xenopus with and without exon 6 it is highly probable that exon 6 is alternatively spliced in all vertebrates. Alternatively spliced isoforms have not been identified in any other species.Dynactin4 from  Figure C. Exon 6Dynactin4 subunits have been predicted to contain N-terminal LIM or RING Vanderwaltozyma polyspora, none of the dynactin subunits were identified, except for dynactin5 and the capping proteins. Vanderwaltozyma also does not encode a cytoplasmic dynein homolog. Thus, the VpDynactin5 might either be an artefact (unlikely) or it gained a species-specific function outside the dynactin complex . The absence of dynein and dynactin in Vanderwaltozyma is most likely related to the specific phenotypic feature that spores are formed by extra mitotic replications after meiosis independent of bud formation[Trypanosoma cruzi. Euglenozoa also contain cytoplasmic dynein heavy and intermediate chains. The presence of only dyanctin5  is in accordance with the report of a freely soluble pool of these subuntis in cells[Vitis vinifera in the cDNA database. This sequence could not be identified in the genome assembly and grouped to a cluster containing parasitic Nematodes in the phylogenetic tree (data not shown) indicating that it is most likely a contamination of the Vitis vinifera cDNA library. Some mammals contain one or more pseudogenes resulting from retro-transcripts.Dynactin5 was found in all eukaryotic branches that contain dynactin, except for species of the Schizosaccharomycetes clade and some yeast species of the Saccharomyces clade. In the yeast species ormation. Sole dyin cells. In addiHomo sapiens is encoded in six exons on chromosome 16 . These contradictions were perplexing and we decided to determine whether potential electrostatic interactions between dynactin subunits and membranes are conserved across the eukaryotes. Therefore, we have analysed the distribution of pI values of the pointed-end complex subunits of all species , plants, Rhizaria, Bacillariophyta, Saccharomycotina and Schizosaccharomycetes. In the Euteleostei branch (containing part of the fish), the dynactin6 gene has been duplicated. The dynactin6 gene from Homo sapiens is located on chromosome 8  and Capβ (Cap2) are part of the dynactin complex but except for capping the Arp1 minifilament they do not seem to have dynactin-specific functions and will therefore be discussed in Additional fileGiardia lamblia), Haptophyceae (e.g. Emiliania huxleyi), Entamoebidae, some of the Microsporidia, and Rhodophyta . While the presence of dynactin is coupled to the presence of a DHC1 there are a few species that contain cytoplasmic dyneins but do not encode dynactin subunits: Piroplasmida (e.g. Babesia and Theileria species), some Microsporidia, and Parabasalia . The DHC1s of the known Piroplasmida and Microsporidia are, however, extremely divergent and shortened  and it is not known whether these are functional motors at all. Together, these results demonstrate the strong functional interconnection between dynactin and cytoplasmic dynein. In addition, both were most probably already present in the last common ancestor of the eukaryotes. Although dynein-independent functions have been reported for dynactin these are most likely sub-functionalisations in specific branches of the eukaryotic tree in which either dyneins partnership became obsolete for certain functions or in which dynactin acquired additional specific binding partners.Here, we have performed an exhaustive analysis of all known dynactin subunits in all eukaryotic genomes available until September 2011. The presence of dynactin subunits is always coupled to the presence of a cytoplasmic dynein heavy chain (DHC1). Some branches do not contain a DHC1 and accordingly do not contain any dynactin subunit Figure A: plantsTetrahymena we were not able to identify dynactin1 homologs in Toxoplasma gondii, Plasmodium and Cryptosporidium species. Therefore, we rather assume that subunits have been lost during evolution although we cannot exclude that we might have missed divergent homologs that can only be revealed in experiments, but not sequence based analyses. In addition, subunits might be missing because of gaps in the sequence assemblies.All of dynactins known eleven subunits were already present in the last common ancestor of the eukaryotes because all of them have been identified in at least two of the major lineages Figure A. HoweveCyanophora paradoxa is not complete, but it seems that based on the latter hypothesis the LECA would have contained only dynactin2, dynactin3, dynactin4, dynactin5, and the CAP proteins. The dynactin data does not help in resolving the issue of unambiguously placing the eukaryotic root because its analysis involves eleven subunits and is biased by the very small number of sequenced species in the taxa Cryptophyta, Haptophyta, Glaucophyta and Excavata except Euglenozoa. Thus it could be possible that more complete dynactin inventories will be found in newly sequenced species of these taxa like in the SAR branch in which all dynactin subunits were found in total but not in a single species. Building a parsimonious tree from the presence and absence of the dynactin subunits alone in all species is not possible without breaking established monophyletic groups like the sistergroups Fungi and Holozoa, or the sistergroups Blastocystis and Oomycetes. However, if we try to reconstruct a tree of the eukaryotes based on the major taxa by only breaking the still debated phylogenetic groupings of the Haptophyta and the Diplomonadida but leaving established supergroups intact, the following scenario can be imagined . Finally, the projecting arm had been completed in SAR and Opisthokonta. However, this model is based on the assumption that dynactin subunits had only been gained and not lost during early eukaryotic evolution, and the model contradicts the unikont-bikont and the photosynthetic-nonphotosynthetic split hypotheses. Given the many dynactin gene loss events in later separating branches it is more likely that the LECA already contained all dynactin subunits. This assumption could be combined with both split hypotheses and is in agreement with analyses of other protein complexes in which the reconstructed complexes of the LECA contained most of the present-day subunits[The evolution of the dynactin complex in eukaryotes is characterised by many branch- and species-specific gene loss and gene duplication events Figure A. The mosubunits,99.Drosophila dynactin1 transcripts without a CAP-Gly domain (corresponding to “p135”) are very well versed to bridge the Arp1 rod to dynein and microtubules showing similar intracellular trafficking of organelles[Drosophila contains an independent Arp1 binding site, and therefore dynactin3 might not be essential for the dynactin complex in all species. This might explain dynactin3’s absence in many branches that have dynactin1. Other reasons could be that we were not able to identify all dynactin3 subunits because of their low sequence conservation or that dynactin3 has diverged in independent branches so far that homology cannot be detected any more. In any case, strong changes happened to this subunit independently in many early branching eukaryotic lineages and also in closely related branches.From the stage of the SAR and Opisthokonta the subsequent evolution of the branches is determined by many and specific gene loss events. Especially Arp10, dynactin6, dynactin1, and dynactin3 have been lost independently in many branches. The Arp1 filament capping function of Arp10 might have been taken over by one of the so far unclassified actin-related proteins or dynactin4. Dynactin6 forms a tight complex with dynactin5 in vertebrates but becaganelles,58,100. ganelles. HoweverDrosophila species[Brachydanio rerio) and in birds, respectively. Arp1 has been duplicated early in vertebrate history and subsequently fish, birds, and amphibians lost different types of the duplicates. An alternative but less likely scenario would be that all dynactin subunit duplications were part of the two whole genome duplications at the origin of the vertebrates followed by numerous independent gene losses in the extant species.Dynactin complex diversity in metazoa is greatly enhanced by branch specific gene duplications and the introduction of alternative splice forms Figure . The dyn species, and in Interestingly, alternatively spliced exons have been invented in vertebrate dynactin1, dynactin2, and dynactin4 genes either before, in between or after the two whole genome duplication events happened but before the divergence of the agnaths and the Gnathostomata Figure . Thus, cBecause most subunits are present in multiple copies in the complex, a single gene duplication of one subunit would already result in two, three, or more different complexes, if complexes were built not only from distinct but also mixed subunit compositions. Based on their gene content, the vertebrates can theoretically build thousands of different dynactin complexes considering all combinations of the genes and splice forms. However, most of the differences are introduced by tiny changes. For example, all identified alternatively spliced exons contain only between two and seven residues. In addition, the two Arp1 paralogs differ in only a few residues that are distributed over the length of the molecule. Thus, these small changes are not expected to considerably alter the overall structure of dynactin. However, it is well known that even single posttranslational modifications can dramatically change the functions of proteins from activating/deactivating enzymes or binding/non-binding other proteins or membranes . ConcerSaccharomyces cerevisiae[Saccharomyces cerevisiae, which does not encode a dynactin5 subunit, that dynein is necessary for nuclear migration and spindle orientation but does not perform vesicle transport[Zygosaccharomyces and Lodderomyces lack Arp10 and dynactin4, the other pointed-end capping protein and it is unclear how the Arp1 filament could be stabilized in these species.In general, gene loss in yeasts only affects the pointed-end complex subunits and dynactin3, which mediates association of dynactin1 to dynactin2 Figure  The Schierevisiae. The dynerevisiae. This isransport,105. In ransport and its The dynactin complex is a very ancient complex that already existed in the last common ancestor of extant eukaryotes. It consists of eleven subunits of which at least seven comprise the core structure: dynactin1, dynactin2, dynactin4, dynactin5, the heterodimeric capping protein, and Arp1. The presence of the dynactin complex coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dyneins like plants and diplomonads also do not encode dynactin subunits either. In the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest repertoire is found in vertebrates. Also at the origin of the vertebrates, several alternatively spliced exons have been invented providing the basis for modulating the core functions. The most prominent example is the dynactin1 gene, from which 36 different transcripts could be generated. In contrast, ascomycetous yeasts have reduced subunit compositions. In general they have reduced pointed-end complexes, which in return let to the loss of the functions coupled to the specific subunits.Dynactin genes have been identified in iterated TBLASTN and PSI-BLAST searches of the completed or almost completed genomes of about 600 organisms starting with the protein sequences of the human dynactin subunits. All hits were manually analysed at the genomic DNA level. The correct coding sequences were identified with the help of multiple sequence alignments of the respective dynactin subunits. As the amount of dynactin sequences increased , many of the initially predicted sequences were reanalysed to correctly identify all exon borders. Where possible, EST data has been analysed to help in the annotation process. In addition to the analysis of these large-scale sequencing projects, all dynactin sequences in the “nr” database at NCBI have been collected and reanalysed.http://www.cymobase.org,[Several of the genes contain alternative splice forms. The different splice forms were not considered independently in the analysis but in all cases the same splice forms were taken for homologous dynactin proteins. All sequence related data  and references to genome sequencing centers are available through the CyMoBase . A listase.org,,109 was The multiple sequence alignments of the dynactin subunits have been built and extended during the process of annotating and assembling new sequences. The initial alignments have been generated from the first about 20 sequences obtained from NCBI using the ClustalW software with standard settings. During Sequences designated “Fragment”, “Partial”, or “Pseudogene” were removed from the multiple sequence alignments of the dynactin subunits. Poorly aligned positions and divergent regions of the alignments were removed using Gblocks with thehttp://www.mbio.ncsu.edu/bioedit/bioedit.html). Shortly, the reported numbers represent the ratio of identities to the length of the longer of the two sequences after positions where both sequences contain a gap are removed. Sequence similarity matrices were calculated with MatGAT[Sequence identity matrices (2D-matrix tables containing sequence identities scores for each pair of sequences) were calculated for each alignment using the method implemented in BioEdit  were substituted by gaps. The phylogenetic trees were generated using two different methods for each dataset: 1. ProtTest was used to determine the most appropriate of the available 112 possible amino acid substitution models[http://www.clcbio.com) and FigTree (http://tree.bio.ed.ac.uk/software/figtree/) and are available as Additional fileFor calculating phylogenetic trees of single dynactin subunits only complete and partial sequences were included in the dataset. For the calculation of the tree of the yeast species, the sequences of the Arp1, Arp10, Cap1, Cap2, and dynactin2 subunit of each species of the Saccharomyces and the Schizosaccharomyces branch were concatenated. Missing protein sequences  subunits showing that the upstream methionines in Neurospora and Sordaria are most likely not the translation start sites.Click here for filePhylogenetic tree of Arp1. The file contains the phylogenetic tree of Arp1 focused on the vertebrate branch highlighting the Arp1 gene duplication event and subsequent branch-specific losses of Arp1 subtypes.Click here for fileDiscussion of the evolution of the Cap proteins. The file contains a discussion of the gene duplications, which happened to the Capα (Cap1) subunit, and of alternative splice variants of the Capβ (Cap2) subunit[ subunit,121.Click here for fileSpecies table. The file contains all species of the analysis, their scientific names, the abbreviation as used in the sequence alignments and trees, the species taxonomy, references to sequencing centers, and publications if genome analyses have already been published.Click here for file


There have been few long-term studies on the outcome of chondrosarcoma and the findings regarding prognostic factors are controversial. We examined a homogeneous group of patients with primary central chondrosarcoma of bone who were treated according to a uniform surgical protocol at our institution, in order to determine the factors that influence survival and identify potential improvements to our therapeutic algorithm. We performed a retrospective analysis of 115 patients with primary central chondrosarcoma of bone who presented with localized disease and who had a minimum follow-up of 5 years after diagnosis. 68 tumors were localized in the extremities and 47 in the axial skeleton or pelvis. 59 patients had a high-grade (II and III) and 56 a low-grade (I) tumor. 94 patients underwent surgical resection with adequate  margins, while 21 patients had inadequate  margins. Tumor grade and localization were found to be statistically significant independent predictors of disease-related deaths in multivariate analysis. The quality of surgical margins did not influence survival. The AJCC staging system was able to predict prognosis in patients with chondrosarcoma of the extremities, but not in those with tumors of the axial skeleton and pelvis. Long-term survival after secondary metastatic disease was only observed when metastases were resected with wide margins. Patients with metastases who received further treatment with conventional chemotherapy, radiotherapy, and/or further surgery had significantly better survival compared to those who received best supportive care. The outcome in patients with primary central chondrosarcoma of bone who present with localized disease is mostly affected by tumor-related parameters. Chondrosarcoma is the second most common primary malignant solid tumor of bone, and accounts for approximately 25% of all bone sarcomas . It is lThere is no consensus on prognostic factors to determine which patients have a higher risk of treatment failure and disease-related deaths, although several papers have addressed this issue . One reaThe purpose of this long-term retrospective study was to examine a group of patients with primary central chondrosarcoma of bone who presented with localized disease and were treated with a uniform surgical protocol at our institution, in order to determine the factors that influence overall and event-free survival. We further aimed at identifying potential improvements to our therapeutic algorithm.146 patients with primary central chondrosarcoma of bone presenting with localized disease were treated at our sarcoma department between 1982 and 2004. 31 patients were excluded from this analysis—6 patients who were primarily treated with palliative intent and 25 patients with follow-up of less than 5 years after diagnosis—leaving 115 patients for this study. At hospital admission, all patients had signed a consent form allowing the use of anonymized information for research purposes.There were 70 male and 45 female patients. The mean age at presentation was 47 (14–79) years and 81 patients were older than 40 years . The mea3.48 tumors were located in the lower extremity, 42 in the pelvic girdle, 20 in the upper extremity or shoulder girdle, and 5 in the axial skeleton. 8 patients presented with a pathological fracture in the lower (n = 5) or upper (n = 3) extremity. Tumor volume was assessed by the pathologist during examination of the surgical specimen. Recorded for 93 patients, it had a mean of 396  cm56 patients (49%) were diagnosed with grade I tumors, 41 patients (36%) with grade II, and 18 patients (15%) with grade III tumors. The grading was based on the system proposed by All patients underwent surgical treatment of the primary tumor. Customized megaprostheses became available at our institution in 1982. Since then, our surgical protocol has undergone only minor alterations, consistently involving the curettage of small, low-grade tumors of the extremities without infiltration of the soft tissue, and a planned wide resection followed by a biological or endoprosthetic reconstruction—when necessary—for all other tumors. Amputations were performed when limb-sparing procedures with wide surgical margins and preservation of a functional limb were deemed impossible, mainly due to tumor infiltration of neurovascular structures or extensive soft tissue infiltration, unless the patient declined to undergo a mutilating procedure.Surgical margins were divided into intralesional, marginal, wide and radical, according to the classification of Enneking. For the purposes of this analysis, intralesional and marginal margins, documented in 21 patients (18%), were characterized as inadequate. Among them were 11 patients in whom a wide resection was initially planned; 4 of these patients underwent a wide re-resection, while 1 patient received adjuvant radiation therapy with 60 Gy. Wide and radical margins, documented in 94 patients (82%), were characterized as adequate surgical margins. 77 patients with tumors of the extremities or pelvic girdle underwent limb-sparing surgery, while 33 patients were amputated.A local recurrence developed in 38 patients (33%) after a mean of 21 (2–96) months. In 2 of these patients, the tumor had dedifferentiated. Distant metastasis developed in 30 patients (26%) after a mean of 27 (2–141) months. 19 patients (17%) developed both a local recurrence and distant metastases at some stage of their illness.Survival analysis was performed using the Kaplan-Meier method. Overall survival was calculated from the date of diagnostic biopsy until death related to disease or treatment, and event-free survival from the date of tumor resection until disease recurrence or death. Survival curves were compared using the log-rank test. Taking into account the different challenges in surgical treatment of tumors of the axial skeleton and pelvic girdle compared to tumors of the extremities, as well as the differences in the clinical course of low- and high-grade tumors, overall and event-free survival were calculated separately for these groups. We performed a multivariate analysis with the Cox proportional hazards model to identify independent predictors of survival in the entire group. The variables entered into this model were the ones previously examined in the literature, in order to permit a direct and consistent comparison of our results with the results of earlier studies. The proportional hazards assumption was checked by plotting the logarithm of the cumulative hazards functions for each covariate, with parallel curves supporting the proportional hazards assumption. The continuous covariate age was categorized with a cutoff at 40 years. All statistical analyses were carried out using the SPSS software version 16.0 . All tests were 2-sided. A p-value of less than 0.05 was considered significant.At the time of this analysis, 73 patients were alive with no evidence of disease. 32 patients had died from disease, 6 patients from treatment-related complications and 4 patients from other causes. Overall survival rates of the entire group at 5 and 10 years were 72% and 69%, respectively. Event-free survival at 5 and 10 years amounted to 57% and 53%, respectively. Both overall  and even3 had a statistically significantly better overall and event-free survival than those with a tumor volume of between 100 cm3 and 200 cm3. The influence of volume on survival was diminished in tumors greater than 100 cm3  = 5, 95% CI: 2–12; p < 0.001) and a tumor localization in the axial skeleton and pelvis .One of the most interesting findings in our series concerns the quality of surgical margins. No effect could be shown on overall survival, both in univariate and in multivariate analysis, although a strong trend of improved survival was found in the AP group for adequate margins. At first, this finding seems to contradict previous studies, which demonstrated that inadequate surgical margins were associated with poorer prognosis . HoweverGiven that the development and subsequent treatment of a local recurrence can lead to increased morbidity and compromise functional outcome, we do not suggest that these results can justify inadequate surgery of otherwise resectable tumors—with the exception of small, low-grade tumors of the extremities without infiltration of the soft tissue. However, we do think that, in light of these findings, the role of ablative surgery in patients with locally advanced tumors of the extremities for which a limb-sparing resection with wide margins is deemed risky should be examined further. This especially so in patients who refuse amputation or in patients for whom a secondary amputation is recommended after inadequate resection of the primary tumor, as there was no correlation between type of surgery and outcome in our patient cohort.3, as it was shown to be an important predictor of overall and event-free survival. With volumetric data missing in 7 out of 47 patients in the AP group, such an alternative staging system correlated better with overall and event-free survival in our series—but also failed to reach statistical significance (data not shown).Another surprising finding was that the AJCC staging system did not correlate with oncological outcome in patients with tumors of the axial skeleton or pelvis. To our knowledge, this has not been reported previously. The reasons for this are unclear. The retrospective nature of our study did not allow an evaluation of the Musculoskeletal Tumor Society (MSTS) staging system, as accurate data regarding tumor compartmentalization were missing in many cases. A dismal prognosis after development of secondary metastatic disease has been reported in many studies . In our 3 of the 56 patients with initially grade I chondrosarcoma developed metastasis and died of their disease within 5 years. Several series examining the course of grade I chondrosarcoma have reported similar results, i.e. a low risk of metastatic disease but high mortality rates after metastasis ranging from 70% to 100% . It appeThe presence of a pathological fracture at diagnosis led to a statistically significant decrease in overall survival in patients with chondrosarcoma of the lower extremity, but not in those with tumors of the upper extremity. In conclusion, the outcome of patients with primary central chondrosarcoma of bone who present with localized disease is mostly affected by tumor- and patient-related parameters. There appear to be subgroups of patients who might benefit from modifications in the standard practice, including those who have traditionally been considered as candidates for ablative surgery, as well as those with distant metastasis who are unwilling or unable to participate in phase II clinical trials. The AJCC staging system does not appear to accurately predict the risk of disease-specific mortality for all subgroups of patients.