RNA from eight Ts65Dn mice (a model of Down syndrome) and eight euploid mice were analysed by real-time PCR to examine inter-individual gene expression levels as a function of trisomy. Down's syndrome (DS), or trisomy 21, is a complex developmental disorder that exhibits many clinical signs that vary in occurrence and severity among patients. The molecular mechanisms responsible for DS have thus far remained elusive. We argue here that normal variation in gene expression in the population contributes to the heterogeneous clinical picture of DS, and we estimated the amplitude of this variation in 50 mouse orthologs of chromosome 21 genes in brain regions of Ts65Dn (a mouse model of DS). We analyzed the RNAs of eight Ts65Dn and eight euploid mice by real-time polymerase chain reaction.App, Cbr1, and Mrps6 exhibited tight regulation in the three tissues and are therefore attractive candidates for further research.In pooled RNAs, we confirmed that trisomic/euploid gene expression ratios were close to 1.5. However, we observed that inter-individual gene expression levels spanned a broad range of values. We identified three categories of genes: genes with expression levels consistently higher in Ts65Dn than in euploids ; genes whose expression levels partially overlap between the two groups ; and genes with intermingled expression, which cannot be used to differentiate trisomics from euploids . Of the genes in the first category, This is the first analysis addressing inter-individual gene expression levels as a function of trisomy. We propose a strategy allowing discrimination between candidates for the constant features of DS and those genes that may contribute to the partially penetrant signs of DS. Down's syndrome (DS) is caused by the presence of an extra copy of chromosome 21 (Hsa21) and is the leading genetic cause of mental retardation in the human population. More than 80 clinical features can occur in DS  that affAnalyses of partial trisomies in DS were instrumental in establishing genotype-phenotype correlations -6; howevIt is reasonable to postulate that changes in expression levels of the genes encoded on Hsa21 are primarily responsible for triggering the pathogenesis observed in trisomy. By analyzing RNAs pooled from several Ts65Dn mice in order to minimize inter-individual variation, we and others demonstrated an overall elevation in expression of approximately 1.5-fold for nearly all transcripts of trisomic genes across multiple tissues -20. BearGiven that changes in gene dosage affect the expression levels of virtually all genes present in three copies, it is reasonable to assume that some of those genes will be neutral for organism fitness, whereas others will exert pathological effects when expression reaches a critical threshold above basal level. However, it is not straightforward to predict which genes will be deleterious when they are over-expressed modestly, even with knowledge of the genes' functions. Among the most pressing questions in DS research are as follows: which genes contribute to specific, constant DS phenotypes (and which do not), and what are the genetic factors that contribute to the phenotypic variability between individuals? Identifying those phenotypes that are associated with differences in gene expression is essential to elucidating the molecular basis of complex traits and disease susceptibility. A recent study  showed tHere, we examine inter-individual differences in expression for 50 mouse orthologs of Hsa21 genes (referred to herein as 'mmu21' genes) in three brain regions of Ts65Dn mice and control littermates (euploids) by means of real-time PCR. This analysis allowed prioritization of the candidate genes for trisomic phenotypes.Transcript levels of mmu21 genes were measured by qPCR in the cerebellum, midbrain, and cortex of eight Ts65Dn and eight control adult mice. Fifty mmu21 genes were tested, of which 33 are triplicated and 17 are disomic in Ts65Dn. Experiments were done in triplicate using two non-mmu21 reference genes that were previously tested for stability in each tissue. For each gene, we calculated a normalized expression value relative to the two reference genes .App, Son, S100b, Itsn, and Dyrk1a), others were differentially expressed . Eight genes  were excluded from further analysis because they had very low or undetectable levels of expression .A large majority of the mmu21 transcripts was found to be active in brain. Forty-two genes (31 trisomic and 11 disomic) were expressed in all three of the brain regions tested. Whereas some of those genes were expressed at high levels in all brain regions . We confirmed a trend of 1.5-fold over-expression for the trisomic genes  . Indeed,We used pooling schemes with eight individual Ts65Dn and control mice as a prerequisite to assess the robustness of our measurements comparing e-pools and b-pools. We validated the 1.5-fold over-expression of trisomic genes in Ts65Dn. However, this value represents a global trend that does not exhibit potential variations in gene expression between individuals. In the next step, we investigated the variation of expression levels for the mmu21 genes in the eight trisomic and control mice.Analysis of individual samples allows recovery of important information that cannot be determined from pooled samples. We estimated the variation of gene expression between individual mice by using the coefficient of variation (CV). We then assessed whether the two populations (Ts65Dn and euploid) differ significantly in terms of this variance by using the F-test, and we applied the Wilcoxon test to judge whether the differences in expression levels between Ts65Dn and euploid animals were significant. Data are summarized in Additional data file 3.It was first important to evaluate the potential influence of technical variation as compared with that of biological variation. We calculated technical variance based on the experimental replicates and biological variance measured between individuals in each group , similar to analysis of variance estimations. We considered the percentage of technical variance over the total variance  as a quality measure. The inter-individual variation in gene expression observed here can be attributed mostly to biologic differences because for most genes the biological variance contributes to more than 90% of the total variance . We could reliably detect expression differences as small as 1.3-fold for nearly all of the assays, because the average standard error of the mean for the relative expression measurements was 0.14 in the 95% confidence intervals.Jam2 in cerebellum; App, in midbrain; and App, Wdr4, S100b, Gabpa, and Mrps6 in cortex). App was previously reported to exhibit highly variable expression in lymphoblastoid cell lines [Kcne2, C21orf7, Cldn8, and Sh3bgr) exhibit a systematic high fluctuation in expression levels (CV > 0.5) across individuals and tissues. Among the disomic genes, two were tightly regulated (CV < 0.1) in cortex (Wdr4 and Lss) and one in the midbrain (Cstb). One disomic gene (Col18a1) exhibited high variation in expression levels (CV > 0.5) among all brain regions and in both groups (Ts65Dn and euploid).We used the CV  as an indicator of the variation in gene expression among individual mice in each group (Ts65Dn or euploid). The CV, in contrast to the variance, is independent of the gene expression level and thus enables inter-gene comparison. To group the CVs, we set arbitrary cut-offs defined as a very low (CV < 0.1), low (CV < 0.2), moderate (CV between 0.2 and 0.5), and high (CV > 0.5) variation in gene expression. In control mice six genes exhibited a CV < 0.1, suggesting that they are tightly regulated .For all of the trisomic genes, we plotted CVs for the Ts65Dn population against the CVs for the euploid population . For most of the tested genes there is no significant difference in the variation of expression levels between trisomic and euploid mice, because 85 out of 93 data points do not differ by more than 20% in CVs between the two groups. On average, 90% of the trisomic genes exhibit low or moderate variation in expression among groups. A few outliers, such as P > 0.05) between euploid and Ts65Dn mice . The basal level of expression of a given gene follows a Gaussian distribution of similar width in trisomic and in euploid mice. This result confirms earlier findings in fetal cortex of human trisomy 21 [P < 0.05) in Ts65Dn than in euploid mice . Contrary to our expectations, however, the CV was significantly smaller (P < 0.05) in trisomic mice for five trisomic genes . Moreover, we see few disomic genes with significant differences in CV (P < 0.05) between the two groups in the cerebellum; for S100b and Ncam2 the CV increases in Ts65Dn mice, whereas it decreases for Lss.The variance in expression levels increases as gene expression increases. This obscures the fact that, in proportion, the level of variation is not greater. To determine statistically whether the presence of an extra copy of a gene influences its variation in expression level, we used the CV calculated with the F-test to circumvent this artefact. We observed that, overall, the amplitude of variation of gene expression did not differ significantly  and Cldn8  confirmed that pooled Ts65Dn/euploid ratios did not reflect a genuine trend toward down-regulation at the level of individual mice. However, it should be noted that this gene, like Cldn8, is expressed at very low levels in brain. Similar effects are observed for these genes in cerebellum and cortex.Finally, we evaluated the significance of the differences in expression levels between Ts65Dn and euploid mice by applying the nonparametric Wilcoxon test, which is robust against outlier values . For instance, in the midbrain the Ts65Dn/euploid ratio values of two trisomic genes are downregulated; these genes are Bace2 in the cortex (×2.15), Kcne2 (×3.39), and Sh3bgr (×0.55) in midbrain [Kcne2, for which the CV values where always above 0.5 in all groups and all tissues. Both b-pool and e-pool ratios for Kcne2 are in the 1.5-fold range in the midbrain, whereas the apparent higher order upregulation in the cortex , does not reflect the individual expression levels spanning the largest range , midbrain , and cortex . For Cbs, a consistent and significant trend toward downregulation  and in midbrain . However, individual gene expression levels for Col18A1 were broadly dispersed in these tissues, and the two groups (Ts65Dn and euploid) are not clearly distinguishable (P > 0.05).Pool data indicated that App in cortex or Mrlp39 in midbrain), enabling a clear distinction between the two populations; genes whose expression levels partially overlap between the two populations of mice ; and genes with intermingled expression between the two populations , which do not distinguish Ts65Dn from euploid animals. As expected, all of the 11 disomic genes tested here were found to be in the latter category except Cbs (see above).Three main categories of trisomic genes can be distinguished from the normalized expression levels: genes whose expression levels in the eight Ts65Dn mice are clearly above those in their euploid littermates . It should be noted that in this first category we observed that, for some genes, one individual out of the trisomic or control group exhibited a slight overlap in its transcript level with that of the second genetic group, although it was still associated with consistent and significantly elevated expression levels in one group of mice as compared with the other  belong to this highly differentiated category for all three brain regions. Furthermore, many genes in this category  are conserved at least in Caenorhabditis elegans and Drosophila melanogaster, and are tightly regulated with a CV below 20% in trisomic or euploid, or both, indicating that these genes are important for development of the organism. Two genes that are good candidates for neurodegenerative pathologies associated with DS are App, which is mutated in some forms of Alzheimer's disease [Itsn (Intersectin), which is involved in clathrin-mediated endocytosis [Jam2 is associated with cell adhesion processes and Cct8 with protein folding and degradation. C21orf5 is a new member of the Dopey family and exhibits restricted regional brain expression [Based on the distribution of expression levels in the brain, the three categories of mmu21 genes at dosage imbalance are presented in Figure  disease  and of w disease  and Itsnocytosis . Jam2 ispression .P values between 0.01 and 0.05). Overall, 22 genes are included in this category. However, of these only two genes are found in all three brain tissues (C21orf51 and Dscr2). In general, genes in this category exhibit moderate variation in expression based on the CV. We hypothesize that genes whose expression is more variable are more likely to contribute to those pathogenic outcomes in DS that exhibit variable expressivity.The second category is populated by trisomic genes for which expression levels are partially or totally intermingled between the two populations of mice (P > 0.05). Overall, 15 genes are included in this last category, and among these five were found in the three tissues . They all have moderate to high expression variation based on the CV. Thus, those genes may be less likely to contribute to constant trisomy-related phenotypes. Genes whose expression levels vary widely among individuals are more likely to reach a critical threshold of over-expression in a subset of individuals, and thus they might be more likely to be involved in phenotypes that occur in some but not all individuals with trisomy 21. We also observed notable behaviour differences between tissues. Four genes  were found in the first category for cortex, but were allocated to the third category for cerebellum. Notably, in midbrain Ets2 was also found in the first category whereas Gabpa, Il10rb, and Tiam1 were found in the second category. Either these genes are tightly controlled in a tissue specific manner or more samples must be analysed to assign them to one or the other group with certainty.The third class includes genes for which the expression levels are intermingled between the two populations of mice  or dramatically different expression levels across individuals. Consequently, Ts65Dn/euploid ratios must be interpreted with caution. For instance, three genes that were previously shown to escape the 1.5× rule, namely Analyses of pooled RNAs minimize inter-individual variations and have been useful in providing an averaged measure of over-expression level in trisomic tissues and to identify possible outliers. Nonetheless, pooling schemes are also associated with intrinsic errors , which may contribute to additional variance. Even though methods have been proposed for accurate analysis of data from large-scale DNA pools , Ts65Dn/As in most expression profiling studies, our data represent a snapshot of the expression level in one individual at the time of death. We cannot exclude the possibility that the expression of some genes may be sensitive to the local environment . Inherent individual variations in the 'personal statistics' of the mouse , all of which affect the number and proportions of cell types in tissues and organs, may lead to changes in the RNA population as well. Some of the variation in expression that we observe may also reflect variation over time or cycling of gene expression levels. However, it is unlikely that cells are synchronized throughout a complex tissue, and such effects are expected to be averaged out for most genes. Ts65Dn is maintained as an advanced intercross between the inbred B6 and C3H strains and thus have some variability in genetic background. We expect that the genetic contribution to differences in expression phenotypes is not large between strains of mice that are relatively closely related (C3H and B6), and euploid/trisomic variation in genetic background is further reduced by using littermate pairs. Nonetheless, allelic variation between individual Ts65Dn mice still represents a factor that must be taken into account. At the same time, variations seen consistently on the more robust genetic background are likely to be representative of 'real' populations. Any or all of these factors may contribute to the observed variation.We, as have others previously , posit tOur results demonstrate the importance of considering gene expression in individuals, and this approach is even more important in human samples, which exhibit greater genetic background heterogeneity than do Ts65Dn mice. Normal variation in gene expression plays a role in susceptibility to complex diseases and likewise plays a potentially relevant role in the phenotypic differences seen between individuals with DS. Although DS presents highly variable clinical features, some phenotypes are common to all, irrespective of genetic background. We expect that these common features derive from dysregulated gene expression that exhibits the same pattern in all individuals. Here we identified three classes of genes with different expression levels relative to euploid. The first class is populated by genes whose expression levels are significantly higher in trisomic than in euploid individuals. The second class is represented by genes with partially overlapping expression levels between the two populations, whereas genes with high degrees of intermingled expression levels form the third class. We postulate that genes in the first class represent good candidates for the constant phenotypes of DS.Cct8 and Ifnar1 appeared to be tightly regulated in cerebellum and cortex. In a study investigating gene expression variation in 40 human lymphoblastoid cells lines [CCT8 and IFNAR1 also appeared to be tightly controlled. This observation shows that, despite the fact that different tissues were investigated, and given that lymphoblastoid cell lines may not always reflect the situation in primary tissues, the use of mouse tissue can be predictive of gene behavior in human. However, we should keep in mind (as discussed above) that even within a given organism, a specific gene may appear tightly regulated in one tissue but not in another. Also, we cannot exclude the possibility that this variation pattern could change during development, but no data are currently available to address this latter issue.In this first, class ls lines , CCT8 anApp, Cbr1, and Mrps6), identifying these as strong candidate genes for Ts65Dn neuroanatomic defects. Along this line, Salehi and colleagues [App (which encodes the amyloid precursor protein) causes abnormal transport of nerve growth factor, resulting in cholinergic neurodegeneration in a mouse model of DS. In contrast, the genes whose trisomic expression levels overlap completely with euploid appear less likely to be key players in the invariant features of trisomy. Among those, we found that Kcne2 and Sh3bgr exhibited a dramatic variation in expression level regardless of ploidy. Expression levels for a number of genes fell between these two extremes, as represented by the second category. This may indicate the limit of precision for this method, but it could also represent a pool of candidates for partially penetrant phenotypes. If the disomic level of a given gene is close to a critical threshold, then elevated gene expression might be deleterious only to those trisomic individuals with the highest expression, contributing to variability in the occurrence of DS features.Three genes from the first category are common to cerebellum, cortex, and midbrain , which are candidates for the trisomy phenotypes. This is a novel dimension in the search of culprit genes for DS that enables one to propose a short list of putative candidates among the genes at dosage imbalance.Total RNA was extracted from frozen tissues of Ts65Dn males and their euploid male littermates using Trizol reagent , following the manufacturer's instructions. Animals were between 13 and 16 weeks old. RNA was treated with RNase-free Dnase I, quantified by UV spectrophotometry, and its integrity was verified by gel electrophoresis. To create the sample pools, equal amounts of RNAs from four euploid or four trisomic animals for each brain tissue were pooled. RNA was transcribed into cDNA using random hexamers and SuperscriptII reverse transcriptase (Invitrogen). In total, 8 μg total RNA for each sample was converted into cDNA in 8 × 1 μg reactions, pooled, and diluted to 12.5 ng/μl equivalent total RNA.® probe and primer sets provided by Applied Biosystems  . All assays met the amplification efficiency criteria of 100% ± 10%  were selected and data were normalized to their geometric mean. All assays were performed in triplicate. To minimize intra-assay variation, the sample cDNA was premixed with the PCR mastermix and distributed equally into each reaction. For a given target gene all tissue samples were run on the same reaction plate. This increases the accuracy of inter-individual comparison, because the mRNA of interest is amplified under the same PCR conditions in all tissue samples. To validate the reproducibility of our system, one experiment including two cDNA samples was duplicated. The correlation between the two independent experiments was in excess of 99% (data not shown).For quantitative gene expression studies, we used pre-designed, gene-specific TaqMan7AP05-02 ) and wer7AP05-02 . Thus, tAll reactions were set up in 10 μl volumes and used the TaqMan Universal PCR Master Mix . Assays were processed with the ABI Prism 7900HT Sequence detection System  under the following conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s/60°C for 1 min. Amplification plot and predicted threshold cycle (Ct) values were obtained with the sequence Detection Software . Further calculations and graphical representations were done using Excel 2000. We verified that no correlation could be found between threshold cycles (Ct) and expression ratios (Ts65Dn/euploid), indicating that there was no systematic biases within our real-time PCR results. Nonetheless, It should be noted that when the Ct value increases above about 32, the standard error also increases, indicating a loss of precision of the replicate measurements.-ΔCt. A given Ts65Dn/euploid ratio was calculated by dividing their respective NE values. For electronic pool (e-pool) calculation, the NE values for all individuals in a given group (Ts65Dn or euploid) were averaged. For the analysis presented here, we considered binned ratios of 0.8 to 1.2 to be neutral (no change in expression), whereas binned values from 1.2 to 2.0 were considered equivalent to 1.5-fold expression change.A common threshold value was chosen for all genes and the baseline was set manually for individual genes. The relative expression calculation method relies on the principle of the comparative Ct method . Ct values were first normalized (ΔCt) to a geometric mean of the two normalization genes and converted to a relative expression quantity (NE) using the formula NE = 2In order to compute variation across a sample, we calculated the coefficient of variation:Here, In order to estimate the technical variance (TechVar) and the biological variance (BioVar) for each gene across the different individuals and the different technical replicate measurements, we used an analysis of variance-like approach:ij is the j-th replicate measurement in the i-th individual, Where I is 8 (the size of the samples group), J is 3 , NEThe total variance (TotVar) is defined by the following equations:P values in the F-test indicate whether there is a significant difference in expression variation.For judging variance differences in the trisomic and euploid samples, we applied an F-test for each gene. This test requires the two samples to have a Gaussian distribution with the same mean value. Therefore, we divided each individual observation from a sample by the mean value of that sample group prior to analysis. Small t-test, the Welch test, and Wilcoxon's rank-sum test. To evaluate differential expression of the genes, P values of Wilcoxon's rank-sum test were preferred as a reference because this test does not depend for its validity on a specific distribution . Furthermore, this test is robust against outlier values in the sample. A recursive function was implemented to calculate the precise P values [For each gene we performed statistical tests based on the replicate signals in experiments with trisomic and euploid samples. Three standard tests were used in parallel: Student's P values .P values from t-test, tu-test, Wilcoxon test, permutation test, and F-test. Additional data file The following data are available with the online version of this paper. Additional data file Provided is a table listing the references of the gene expression assay (Applied Biosystems) that were used for quantitative RT-PCR experiments.Click here for fileProvided is a figure of the correlation plot of intensities from electronic pools (y-axis) versus biologic pools (x-axis) for each gene in the three brain tissues.Click here for fileP values from t-test, tu-test, Wilcoxon test, permutation test, and F-test.Provided is a summary table listing the following information for each gene and each brain tissue analyzed: gene names (triplicated genes in Ts65Dn are in red), mean expressions (ME) of Ts65Dn and euploid mice, standard errors of MEs, CVs of the Ts65Dn and euploid samples, technical and biologic variance, mean trisomic:euploid gene expression ratios from electronic and RNA pools, and Click here for fileProvided is a scatter plot of CVs of euploid versus Ts65Dn mice in brain tissues. The dotted lines represent the ± 20% CV deviations from the ideal correlation (plain line).Click here for file


Preconception counselling (PCC) can reduce adverse pregnancy outcome by addressing risk factors prior to pregnancy. This study explores whether anxiety is induced in women either by the offer of PCC or by participation with GP-initiated PCC.Randomised trial of usual care versus GP-initiated PCC for women aged 18–40, in 54 GP practices in the Netherlands. Women completed the six-item Spielberger State Trait Anxiety Inventory (STAI) before PCC (STAI-1) and after (STAI-2). After pregnancy women completed a STAI focusing on the first trimester of pregnancy (STAI-3).The mean STAI-1-score (n = 466) was 36.4 (95% CI 35.4 – 37.3). Following PCC there was an average decrease of 3.6 points in anxiety-levels . Mean scores of the STAI-3 were 38.5 (95% CI 37.7 – 39.3) in the control group (n = 1090) and 38.7 (95% CI 37.9 – 39.5) in the intervention group (n = 1186).PCC from one's own GP reduced anxiety after participation, without leading to an increase in anxiety among the intervention group during pregnancy. We therefore conclude that GPs can offer PCC to the general population without fear of causing anxiety.Trial Registration: ISRCTN53942912 By addressing pregnancy-related risk factors and preventive measures before pregnancy, preconception counselling (PCC) reduces the number of adverse pregnancy outcomes . CurrentLittle is known about women's perception of PCC, or whether it might have any adverse effects. Various studies in genetic testing have reviewed the effect on anxiety and reported no long term adverse psychological effects. However, these tests concerned people at high-risk of cancer or hereditary diseases,5 or higThe fact that merely everyone in the Netherlands is registered with a general practice provides an ideal opportunity to reach couples with a low-risk profile at an appropriate time for PCC. As part of an intervention study, 'Parents to Be', we developed a systematic PCC programme specifically designed for GPs to offer to women of childbearing age . The maiTo determine whether the offer of PCC actually induced anxiety during pregnancy, we compared the anxiety levels of a group of women who became pregnant after they had been offered PCC with those of a group who were not.A total of 110 GPs were approached and 67 GPs of 54 practices agreed to take part in the trial. The participating general practices were divided in strata according to the characteristics both of the practice and of the GPs . Within these strata, practices were randomised (by computer) either to the intervention group or to the control group, resulting in 27 practices (30 GPs) in the intervention group and 27 practices (37 GPs) in the control group. Comparable numbers of patients were registered at the intervention and control practices. The power calculation for the main trial was based on an absolute risk reduction of 4% in adverse pregnancy outcome between these two groups of patients. We assumed that 30 pregnancies would occur in a practice each year.Prior to the start of the trial, GPs in the intervention group received training on how to provide PCC. Subsequently, all women 18–40 years of age were selected in the intervention group. Women were excluded if they had undergone hysterectomy or sterilisation, if they were known to be subfertile or infertile, if they had an insufficient knowledge of the Dutch language, if they were difficult social circumstances in their lives or if the GP presumed they had completed their family.The remaining women received a letter explaining the study procedure and were invited to attend PCC. The selection and the offer of PCC took place in 2000, 2001 and 2002 . Women wAt the end of the risk assessment questionnaire prior to PCC, women were asked to fill in the Dutch version  of the sParticipants were asked a number of questions about their perception of their own risk of having a child with a congenital disorder, and how they judged their own knowledge of preventive measures for such disorders. The questionnaire was subsequently returned to the GP in time to prepare for the counselling session.During the session, the GP discussed the individual risk factors of both partners on the basis of the risk assessment as well as general risk factors. Issues that were discussed were genetic counselling, obstetric risk factors, infection prevention, medication use, folic acid use, intoxicants , nutrition and occupational hazards. Immediately after the PCC session, women were asked to fill in another STAI form (STAI-2).All women in both the control and intervention practices who had been pregnant between April 2000 and April 2003 received a questionnaire two months after their pregnancy had ended. GPs excluded women if taking part in the study was thought to be too burdensome due to emotional problems. Lifestyle and behaviour before and during pregnancy, as well as complications and pregnancy outcome were evaluated. At the end of the questionnaire, women were asked to complete the STAI on the basis of their memory of the first trimester of their pregnancy (STAI-3).The medical ethics committee of Leiden University Medical Center approved questionnaires and study.The analyses for STAI-1 and STAI-2 were performed using SPSS 11.5. Risk factors for adverse pregnancy outcomes reported in the risk-assessment questionnaire were divided into three categories. Category I comprised risk factors for which written information would be sufficient, category II comprised risk factors for which personal counselling by the GP was indicated , and category III comprised risk factors for which a referral to a specialist was necessary. Using paired t-tests, we compared anxiety levels prior to the PCC session (STAI-1) with levels after PCC (STAI-2). Using one-way analysis of variance, with trend test -if appropriate- we related background characteristics, the women's perception of risk, and the number of risk factors in categories II and III to the baseline STAI and the change in STAI after PCC .The anxiety levels (STAI-3) of women who were offered PCC were compared with those of women not offered PCC, as well as with those women who had accepted the offer and those who had not. A three-level model was used with questionnaire as first level, women as second level and GP-practice as third level to compare STAI-3 between the groups. We chose to do so, because of the required cluster randomisation and due to the fact that some women had been pregnant more than once during the study period. This model was fitted using Proc Mixed in SAS version 9.1.3Subgroup analyses were performed for age, education, country of origin and adverse pregnancy outcome, the latter being defined as miscarriage, preterm birth, stillbirth or congenital disorder of the newborn.In the intervention group, the risk-assessment questionnaire was completed and returned by 481 women, 466 of whom completed the first STAI in full. Participation and response is described in figure Of the 353 women who actually attended PCC, 223 completed STAI-2 after PCC. The other 130 STAI-2 forms were either not handed out by the GP or were not returned.The mean STAI-1 score filled in before PCC was 36.4  35.4 – 37.3), and showed no difference between women who did and who did not actually attend PCC. Table Higher baseline scores were found among women with a basic education (39.2) and women of non-Dutch origin (41.0). Women who had been pregnant before had a higher mean STAI-1 score (38.0) than those who had not been pregnant (35.6). This was partially accounted for by the anxiety of the women who had a previous adverse pregnancy outcome (39.8).Higher anxiety levels were found in women who believed they had a higher risk than others of bearing a child with a congenital disorder (40.6) or those who perceived their subjective risk as high 42.2) (Table .2 (TableAnxiety was significantly increased in women who felt they had little knowledge of preventive measures (test for trend p = 0.006). There was an association between the number of actual risk factors for adverse pregnancy outcome in category II, which required personal counselling, and the mean STAI-1 score . No significant correlation was found with category III items, which entailed referral to a specialist .Comparison of anxiety levels prior to PCC with the levels afterwards showed an average decrease of 3.6 points . No difference was found in the mean STAI-1 scores between women who did or did not fill in the STAI-2. Neither was a correlation found between the time interval between completing the first two questionnaires and the decrease in anxiety score . The highest reduction in anxiety scores was found among women with a basic education  and women born in countries outside the Netherlands . Relatively speaking, the reduction in anxiety was also larger in women who estimated that they had a high chance of having a child with a congenital disorder . Women who knew little of preventive measures were initially anxious and showed a substantial decline of 5.2 (95% CI 2.2 – 8.2). After PCC there was a smaller decrease in anxiety in women with the highest number of category II risk factors and in those women with serious (category III) risk factors (2.8 respectively 2.0).During the study, 4,062 pregnancies occurred. Anxiety was measured following pregnancy on the basis of women's memory of the first trimester of their pregnancy (STAI-3). Eligibility, exclusion and response to the post-pregnancy questionnaire is described in figure In total, 3,406 (84%) women received the questionnaire and 2,413 returned it (response 71%). 2,276 questionnaires completed adequately were analysed; 1,186 from the intervention group and 1,090 from the control group. Although the age distribution was comparable to that of women of childbearing-age in the general population, the respondents had a relatively higher educational level.As shown in Table Preconception counselling provided by the participants' GP did not induce anxiety, nor did the invitations to women of childbearing age to participate in PCC resulted in higher pregnancy-related anxiety levels. The women who benefited most from PCC were those of non-Dutch origin with a basic educational level, those perceiving their subjective risk as high or those with a low estimation of their own knowledge of preventive measures: these groups showed a substantial decline in anxiety after the PCC session.Although we didn't reach the number of participants we needed for conclusions on adverse pregnancy, we believe that the strength of this study lies in the large number of participants from the overall population of women of child-bearing age. Previous studies on genetic testing,,5 screenWe considered the possibility of selection bias. Our respondents were relatively well-educated compared to the general population. As women could self-select if they were interested, understandably more often women attended PCC who had never been pregnant before or who had experienced an adverse pregnancy outcome. Another bias could be that GPs were able to exclude women if they believed the questionnaire to be too burdensome emotionally. For this reason fewer women with an adverse pregnancy outcome may have received it, thus leading to a selection bias. For example, 9% of all the pregnancies in the study ended in miscarriage , but only 6% of the women who completed the STAI-3 had suffered a miscarriage.There was a difference between the selection of the intervention and control GPs. Probably due to the repetitive selections for the study, the intervention GPs had become more cautious in selecting women, allowing only 46% of the women with an adverse outcome to receive the pregnancy-related questionnaire; with control GPs, the figure was 73%. Of the women who had experienced an adverse outcome, 76% in the intervention group returned the questionnaire versus 70% in the control group. This may have led the actual anxiety scores in the intervention group to be underestimated. However, subgroup-analyses show that there was no difference in mean STAI-3 scores between the women in the intervention and control groups who had had an adverse outcome.The retrospective nature of the STAI-3 is debatable. However, this assessment gave us the opportunity to study anxiety among women who had declined PCA and had had an adverse pregnancy outcome. Prospective assessment of the STAI-3 in this group of decliners, e.g. in the first trimester could have acted as an extra reminder to adhere to risk-reducing behaviour, which would have biased the results.Just like Spielberger who founA supplementary questionnaire was used for a subgroup of our sample to assess women's satisfaction with PCC. In 2003 a small group of 25 women participating consecutively in ten different practices received it two weeks after attending PCC. As satisfaction with counselling may be influenced by overall satisfaction with the counsellor, this was measured as well. In totaOur baseline STAI-score (36.4) was comparable to normative mean STAI score (35.2) and to tThe only significant difference in STAI-3 was in the age group 25–30 years where the intervention group was slightly more anxious. This effect was not observed in any of the other age groups and could therefore be due to the fact that we performed multiple hypothesis tests.Among the women who went for PCC, the mean STAI-3 score relating to the first trimester was 37.8, which did not differ much from the first trimester score of 24 women at the fertility-clinic in Nijmegen (35.7) . While tIn the debate on how to provide PCC, some people wish to concentrate efforts on women who have never been pregnant before. This is understandable, as knowledge of preventive measures is mostly transferred during pregnancy . It may be unwise however: women in our study who had previously been pregnant tended to have higher baseline anxiety levels. If such women were excluded, it would not be possible to address them.To reach women in time for PCC it is essential to enhance the awareness of both women and physicians to risk factors in everyday life that need adjusting prior to pregnancy. Yet, until PCC is incorporated as a normal part of pregnancy care, extending a  invitation seems necessary. To do so physicians or other health care workers will need  support. As in the Netherlands, registration at a GP's practice, will facilitate the process of reaching women but it is not imperative.Our study did not show that PCC induced anxiety when it was offered to women in the general population by their own GP. On the contrary: after the counselling session, the anxiety score was lower. There is no evidence that even those women who had had an adverse pregnancy outcome after declining further participation in PCC suffered any anxiety by the offer of PCC. We therefore conclude that GPs can offer PCC to the general population without fear of causing anxiety.The author(s) declare that they have no competing interests.LCJ was involved in the study design, enrolled participants, monitored data collection, analysed data and drafted the manuscript. JE enrolled participants, monitored data collection and revised the draft. SC was involved in the study design, analysed data and prepared the draft manuscript. KMP was involved in the study design, revised the draft and supervised the project. AKN analysed data and prepared the draft manuscript. SEB revised the draft and supervised the project. WJJA analysed data, prepared the draft manuscript and supervised the project. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Most cervical cancer patients with pelvic recurrent or persistent disease are not candidates for exenteration, therefore, they only receive palliative chemotherapy. Here we report the results of a novel treatment modality for these patients pre-exenterative chemotherapy- under the rational that the shrinking of the pelvic tumor would allow its resection.Patients with recurrent or persistent disease and no evidence of systemic disease, considered not be candidates for pelvic exenteration because of the extent of pelvic tumor, received 3-courses of platinum-based chemotherapy. Response was evaluated by CT scan and bimanual pelvic examination; however the decision to perform exenteration relied on the physical findings. Toxicity to chemotherapy was evaluated with standard criteria. Survival was analyzed with the Kaplan-Meier method.Seventeen patients were studied. The median number of chemotherapy courses was 4. There were 9 patients who responded to chemotherapy, evaluated by bimanual examination and underwent pelvic exenteration. Four of them had pathological complete response. Eight patients did not respond and were not subjected to surgery. One patient died due to exenteration complications. At a median follow-up of 11 months, the median survival for the whole group was 11 months, 3 months in the non-operated and 32 months in those subjected to exenteration.Pre-exenterative chemotherapy is an alternative for cervical cancer patients that are no candidates for exenteration because of the extent of the pelvic disease. Its place in the management of recurrent disease needs to be investigated in randomized studies, however, its value for offering long-term survival in some of these patients with no other option than palliative care must be stressed. Cervical cancer continues to be an important health burden with a yearly incidence of almost half a million new cases in the world and a mortality rate of about 50% . Currentchemoradiation if the primary disease was approached with surgery; b) palliative chemotherapy if recurrence is considered unresectable and the primary disease was treated with radiation or chemoradiation; and c) pelvic exenteration for selected cases with small, central disease even if primarily treated with chemoradiation or radiation. Pelvic exenteration involves en bloc resection of bladder, genital tract, and rectum; it was first described by Brunschwig in 1948 [Recurrent disease can be treated by a)  in 1948 . This pr in 1948  and it iDue to these facts, the vast majority of recurrent cervical cancer patients are left with no curative options, therefore it is important to search for other therapeutic alternatives in patients that are not "standardly" considered for the classical exenterative procedure. The introduction of high-dose-rate intraoperative radiation therapy (HDR-IORT) combined with radical surgical resection has widened the scope of patients who may be offered surgery , howeverHokel et al., have recently described the laterally extended endopelvic resection (LEER) as a novel surgical salvage therapy to a selected subset of patients with locally advanced and recurrent cervical carcinoma involving the pelvic side wall. This consists in extending the lateral resection plane of pelvic exenteration to the medial aspects of the lumbosacral plexus, sacrospinous ligament, acetabulum, and obturator membrane to allow for resection with disease-free margins . With thCurrently, a combination of cisplatin and paclitaxel has shown better response rate and progression- free survival than single agent cisplatin hence, combination chemotherapy as been regarded as the standard of care in patients to be treated with systemic palliative chemotherapy . Chemoth3, and a platelet count of at least 100 000/mm3, total bilirubin less than 1.5 times the normal upper limit (NUL), transaminases less than 1.5 times NUL, and normal levels of creatinine in serum; a normal posteroanterior chest X-ray as well as having the correspondent informed consent. The exclusion criteria included: severe systemic or uncontrolled disease  that precluded the use of chemotherapy and further exenteration; concomitant treatment with any other experimental drug; mental illness and previous or concomitant malignancies except non-melanoma skin cancer. The study was approved by the Institutional Regulatory Boards.All patients had histologically proven persistent or recurrent cervical carcinoma to primary radiation or chemoradiation. At the pelvic examination -under no anesthesia-, these patients were felt to have pelvic fibrosis and diagnosed as having a recurrence of diffuse infiltrative growth pattern (with or without a dominant mass) fixed or not to one or both pelvic side walls. Consequently, these patients were considered by the gynecologist team of our Institution  to be unsuitable for pelvic exenteration regardless of the CT scan findings. Patients also had to meet the following inclusion criteria: Aged between 18 and 70 years; ECOG performance 0–1; adequate hematological, hepatic and renal functions as determined by: hemoglobin equal or higher than 10 g/L, leukocyte count higher than 4000/mm2, d1 and gemcitabine 800 mg/m2 d1&8 (2 patients); carboplatin AUC 5–6, d1, and paclitaxel 135 mg/m2, d1 (3 patients); carboplatin AUC 5–6, d1, and 5FU 1 gr/m2 d1-5, (3 patients); cisplatin 100 mg/m2 d1, and 5FU 1 g/m2 d1-5, (4 patients); and cisplatin 100 mg/m2 d1 and gemcitabine 1 g/m2 d1&d8 (5 patients). Courses were administered every three weeks for a maximum of 6 courses. Conventional antiemetic therapy and ancillary medications were used during drug treatment. Chemotherapy was stopped in cases of disease progression or prohibitive toxicity.Chemotherapy was administered in an outpatient setting. Diverse chemotherapy schedules based on cisplatin or carboplatin were used as follows: Carboplatin AUC 5, d1, paclitaxel 135 mg/mObjective evaluation of response to chemotherapy using standard response criteria was not the primary objective of this study as this would have required that all patients had a well-defined and measurable mass. Instead a response to chemotherapy was defined when the pelvic disease was felt less fixed and/or the "fibrosis" was felt softer by the same team of gynecologists that performed the pre-chemotherapy evaluation. Toxicity to chemotherapy was evaluated according to the NCI Common Toxicity Criteria.After pre-exenterative chemotherapy, patients were evaluated by the same team of gynecologists through pelvic examination (CT scan was not mandatory). However, the decision to proceed or not with the surgical procedure relied only on pelvic examination and was based on whether there was or not response as evaluated with above described criteria. The other criterion for no exenteration was a worsening of the general clinical condition of the patient regardless of the pelvic examination. Patients were followed every three months after completion of all treatment.Overall survival was evaluated using the Kaplan-Meier method and was considered from the date of the diagnosis of the persistent or recurrent disease until the date of death of last visit.From May 1999 to March 2003, 17 patients were studied in this pilot trial. Baseline characteristics of patients (at diagnosis of their primary disease) are shown in Table All patients had histological confirmation of their recurrent disease. The clinical status at entering the study is shown in Table Table Chemotherapy was well tolerated. The most common side effect were nausea/vomiting grade 1 and 2, mild to moderate anemia was present in half of patients; all patients presented leukopenia and neutropenia which were grade 3 in five and three patients respectively. There were no episodes of infection or bleeding Table .In regard to the exenterative procedure, a total infraelevator exenteration was done in eight cases and one had anterior supraelevator exenteration. This patient was the one with positive surgical margins in the vaginal border. It is worth mentioning that, in this case, the transoperative frozen section of the vaginal margin was reported negative; however, the definitive histological analysis showed disease. The definitive histological analysis of the surgical specimens showed a complete pathological response in 4 cases, a residual disease ≤2 cm in four cases, and one case with a residual measuring 8 cm. In seven patients, the urinary diversion consisted of an ileocolonic conduit and an ileal conduit in two cases. Colostomy was done in the eight cases undergoing total exenteration, Table . RegardiAll patients not subjected to exenteration showed disease progression and died within the ensuing months, being the median survival of only 3 months. The status of the operated patients is as follows: patient 1: Path CR, alive without disease at 62 months, patient 2: Path CR, alive without disease 59 months; patient 3: Residual of 2 cm, local and regional recurrence at 7 months post-exenteration, patient 4: Residual of 2 cm, alive without disease 52 months, patient 5: residual of 2 cm, local recurrence at 10 months, patient 6: residual of 2 cm, local recurrence at 7 months, patient 7: Path CR died at four months from surgical complications, patient 8: Path CR, died at 20 months from liver recurrence; patient 9: Residual of 8 cm, alive without disease 13 months. Thus, four of the nine operated patients are alive without disease. Median survival in the intention to treat was 11 months, being 3 versus 32 in the non-operated versus those that underwent exenteration  out of the 17 patients included in this trial underwent pelvic exenteration obtaining disease-free margins in all but one case; four of them are alive without disease.The clinical characteristics of the patients included in this study are remarkable in the sense that all of them were considered no suitable for pelvic exenteration according to standard criteria by the team of gynecologists of our Institution. This special subgroup of patients with recurrent disease is better defined if we look at their clinical characteristics: 13 of them relapsed at a median time of only 16 months whereas four were refractory to primary treatment and progressed within two to seven months All of them complained of pelvic pain, five had unilateral leg edema, three presented hydronephrosis and three cases had both signs. Both the short disease-free interval and the presence of one or more of the typical triad of signs and symptoms are either contraindications or factors predicting a very poor outcome after exenteration in most of the reported series -20.Selecting the true candidates for pelvic exenteration is a difficult clinical dilemma in patients with recurrent cervical cancer after radiation therapy. Despite very thorough preoperative investigation, inoperable disease is discovered at the time of laparotomy in up to 50% of cases . CT scanIt must be stressed, however, that all these imaging and laparoscopy efforts to predict resectability and avoid aborted exenterations are done in the setting of "classic indications" of pelvic exenterations, where the ultimate goals are increasing the efficacy of the procedure in terms of disease control and decreased morbidity and mortality. However, our approach in aimed at increasing the proportion of patients in whom this salvage therapy could be attempted under the rationale that systemic chemotherapy would allow obtaining free surgical margins in situations where the extent of pelvic disease predicts that negative surgical margins would unlikely be obtained and therefore exenteration could not be offered to these patients.These considerations led us to rely on bimanual pelvic examination, which is a subjective test, as our principal criterion for deciding to perform the exenteration . We acknowledge that it would had been very valuable to have an objective pre and post-chemotherapy evaluation of the response by a CT scan, RMI, and/or PET scan in all the cases, however, only nine cases had CT scan pre and post therapy. Notwithstanding it is interesting to notice that although all our patients were "felt" by physical exam to have side wall fixation, the CT scan confirmed it only in six cases based on the criterion of having a less than 3 mm separation of the tumor from the pelvic muscles and/or vascular encasement . It is iNevertheless, an important observation is the fact that in three out of the four cases that achieved a pathological complete response, the CT scan was clearly positive for the presence of tumor. This is shown in figure The use of chemotherapy in the palliative setting of persistent or recurrent pelvic disease, particularly in a patient who has received definitive radiation or chemoradiation treatment has very limited value. In a review on results of 190 advanced or recurrent disease patients treated with 14 different chemotherapy protocols, the overall response rate was 20.0% , with a median response duration of 4.8 months . In a reSo far there is information of the efficacy of chemotherapy in terms of pathological response in recurrent or advanced cervical cancer because chemotherapy is only used as a palliative measure and surgery is commonly not affered after chemotherapy. Here we demonstrate a pathological complete response rate of 44% in the nine patients treated (23.5% taking into the 17 patients), which is higher than obtained in neoadjuvant trials in locally advanced cervical cancer utilizing platinum-based schemes with newer drugs, such as gemcitabine ,33, vinoA noticeable finding of the present report is that the median survival of 11 months compares favorably with studies using systemic chemotherapy in the palliative setting, ranging from 6 to 10 months ,39; howeThe therapeutic modality here reported, called pre-exenterative chemotherapy, is a therapeutic alternative for cervical cancer patients with recurrent or persistent disease limited to the pelvis not usually considered candidates for "classical" pelvic exenteration. Its value in the management of recurrent disease needs to be confirmed in a randomized phase III study.The author(s) declare that they have no competing interests.R D-V, J U-D, JC and RB participated in data collection and analysis; LC, DC, C L-G, A G-E and A D-G managed the patients; MC, and J de la G critically read and participated in the manuscript; additionally C L-G and A D-G conceived and wrote the manuscript.The pre-publication history for this paper can be accessed here:


Pulmonary vein (PV) isolation (PVI) techniques have evolved as a curative treatment of atrial fibrillation (AF) since PVI guided by circumferential mapping with a circular catheter was initially proposed. A multielectrode basket catheter (MBC) is also useful for PVI because of some advantages; (1) an MBC provides some information about the PV anatomy on the fluoroscopic image, (2) an MBC can utilize the non-fluoroscopic navigation system, (3) an MBC enables the direct three-dimensional mapping around the PV ostium and antrum, (4) the distal electrodes of the MBC can be used to monitor some activation changes within the PV in real time and thereby indicate the effects of ablation at the ostium and antrum as radiofrequency lesions are created. PVI with an MBC is an effective and safe procedure to cure AF by integrating the PV anatomy and electrophysiology in combination with a non-fluoroscopic three-dimensional navigation system for the ablation catheter. The pulmonary veins (PVs) have been demonstrated to be the major source of triggers of atrial fibrillation (AF) . Since PThe MBC  consisted of eight flexible, self-expanding splines (A-H) with eight 1-mm electrodes with 2 or 3-mm (31 or 38-mm MBC) spacing . A speciA computerized three-dimensional (3-D) mapping system (QMS2™) could construct a 3-D color isochronal or isopotential map from a total of 56 bipolar electrograms recorded by the MBC. The electrical activity in the space between the splines was estimated by a Bicubic-Spline interpolation to construct a continuous map. A color setup with a gradation which corresponded to the relative time or amplitude of the potential could be arranged variously on the QMS map.A non-fluoroscopic navigation system (Astronomer™) associated with the MBC could be utilized for mapping . This syPrior to the introduction of the MBC within the PVs, a selective PV angiography was performed to evaluate the diameter of the PV and distal branching pattern . A guidiHaissaguerre et al. demonstrated that circumferential PV ostial mapping with a circular catheter could identify a segmental electrical connection between the PV and LA and a successful PVI could be achieved by radiofrequency (RF) ablation targeting those electrical disconnections . The MBCPV mapping with a 3-D mapping system (QMS2™) was useful for identifying a preferential electrical connection and determining its elimination accurately because it enabled not only a visualization of the activation sequence of the PV potentials but also an adequate evaluation of the activation sequence between the splines ,9. GenerAn MBC has several advantages over a circular catheter in PV mapping ,6-8. FirOn the other hand, an MBC has several disadvantages over a circular catheter ,6-8. FirThe AF recurrence rate after the first procedure in the paroxysmal AF patients was 72% and 51% in Kumagai et al.'s and our report, respectively. The AF recurrence rate after multiple procedures was 70 to 80% in the paroxysmal AF patients ,7,8. TheThe incidence of significant PV stenosis (> 50%) was reduced remarkably (0 to 1.2%) ,7,8 withBecause circumferential PV ablation by an anatomical approach has achieved a better clinical result  than segArentz et al. developed circumferential ostial PVI with an MBC . In theiAfter the circumferential ablation, 49% of the PVs had been successfully isolated. In all the remaining PVs with residual conduction gaps, complete PV electrical disconnection could be achieved by an electrophysiologic isolation technique.In general, the PV antrum is defined as the proximal portion next to the tubular PV as observed on the selective PV angiogram or intracardiac echocardiogram ,13. It hAfter the circumferential PV antrum ablation, PV antrum isolation was achieved in 77% and a residual PV conduction gap through the previous ablation line was observed in 23% of all the targeted PVs. All of the residual PV antrum conduction gaps were eliminated by a few local RF deliveries and the electrical disconnection of all targeted PV antra was successfully completed.The AF recurrence rate after the first procedure was 62% and 84% in Arentz et al.'s   and our Although the splines of the MBC are coated with heparin, the geometry of the MBC is more complex than a circular catheter, a factor that may predispose to greater thrombogenicity. Carbonization can sometimes be observed after ablation on the MBC electrodes or splines. Carbonization is thought to be caused by the concentration of RF energy on the thin splines, which results in very high local temperatures that induce denaturalization of proteins. In spite of those factors, the incidence of thromboembolic events was very low (0.5%) ,8,12,13.No pericardial tamponade associated with an MBC have been reported in any of the literature -8,12,13.Over the last decade, considerable advances have been made in the catheter ablation of AF. The success rates have improved with the evolution and development of PV isolation ,15. HoweOur electrophysiologic PV antrum isolation with an MBC could eliminate AF without any critical complications in about 90% of the paroxysmal AF patients probably because of achievement of a high occurrence of permanent complete PV electrical disconnection and covering non-PV foci. Therefore, we think that our minimally extensive PV antrum isolation technique can target the optimal sites in order to achieve both a high efficacy and safety. Though our results are preliminary and must be confirmed by further studies with a larger number of patients and longer follow-up, our PV antrum isolation technique seems to be the best of the previous PVIs with an MBC.The mechanisms of initiating, maintaining, or perpetuating AF are different, complex, and certainly of multiple kinds especially in persistent or permanent AF, and it is reasonable to believe that they cannot be eliminated altogether by PV or PV antrum isolation alone . It was PVI with an MBC is an effective and safe procedure to cure AF patients by integrating the PV anatomy and electrophysiology in combination with a non-fluoroscopic 3-D navigation system for the ablation catheter.


Altered spinal mobility is thought to be related to current or past episodes of low back pain; however evidence of that relationship in younger subjects has not been established. The purpose of this study was to compare lumbar segmental mobility in asymptomatic and symptomatic subjects during posterior to anterior (PA) manual spinal mobilization and a self-initiated prone press-up (PU) maneuver. We hypothesized that persons with central low back pain would have an altered lumbar segmental mobility pattern compared to those without pain.Forty-five individuals (age 32.1 ± 8.5) with non-specific low back pain and 20 persons (age 31.1 ± 7.0) without low back pain participated. Each subject underwent dynamic imaging of the lumbar spine during a PA mobilization procedure and while performing a PU. Segmental motion was quantified as the change in the intervertebral angle between the resting and end-range vertebral positions.The symptomatic group had a larger percentage of subjects with evidence of single level segmental hypermobility than the asymptomatic group during the PA (40.0% vs. 5%) and PU (26.7% vs. 15%) procedures. Single lumbar motion-segment analysis revealed hyper-mobility in symptomatic subjects at L5 – S1  and L4 – L5  during the PA test.Persons with non-specific low back pain have a tendency to demonstrate single level lumbar segmental hypermobility when compared to age specific asymptomatic subjects. Back pain and its association with altered functional ability can significantly affect an individual's quality of life ,2. PrevaA painful low back does not indicate a specific pathology but may be related to restricted, excessive, or poorly controlled lumbar motion -12 AlterGeneral trunk mobility has been quantified in the clinical setting using methods of linear or angular displacement . These tIt is not clear if persons with current back pain, or those with a history of frequent episodes of back pain, have altered segmental spinal mobility. Previous studies have attempted to correlate spinal mobility to clinical symptoms but have not been able to clearly establish a relationship -22. StruQuantity and pattern of segmental mobility may certainly vary with the type of movement tested, instructions to the subjects and their willingness to participate. For example, a self initiated test, such as a press-up (PU) maneuver may be influenced by the subject's symptoms, thereby limiting spinal segmental motion. In contrast, application of a manual PA force to a lumbar spinous process sufficient in amount to reach end-range may produce more motion since it's not self-limited by pain. Therefore, quantifying motion under these two conditions may provide a more comprehensive assessment of segmental mobility in persons with low back pain. Segmental motion of the lumbar spine during manual PA assessment has been investigated -27 and wUsing dynamic imaging techniques, the purpose of this study was to compare lumbar segmental mobility in asymptomatic and symptomatic subjects during PA spinal mobilization and a prone PU maneuver. We hypothesized that persons with central low back pain would have an altered lumbar segmental mobility pattern compared to those without pain.A total of 65 individuals between the ages of 18 and 42 participated in this study. The asymptomatic group was comprised of 20 healthy individuals, with no history of low back pain lasting more than 3 days or pain reported within the previous year (Table The primary exclusion criteria for both groups were: 1) spinal malignancy, 2) cardiovascular disease, 3) evidence of cord compression, 4) aortic aneurysm, 5) hiatal hernia, 6) uncontrolled hypertension, 7) spinal infection, 8) severe respiratory disease, 9) pregnancy, 10) abdominal hernia, 11) prior low back surgery, 12) gross spinal deformity, 13) spondylolisthesis, 14) known rheumatic joint disease, and 15) implanted biological devices that could interact with the magnetic field . In addition to the above exclusion criteria, subjects in either group could not have any signs or symptoms related to nerve root or cord pathology. Therefore, subjects who demonstrated any of the following also were excluded: 1) radiating pain below the level of the buttock(s), 2) sensation changes in the lower extremities, 3) diminished reflexes, 4) lower extremity weakness, 5) urinary or fecal incontinence, and 6) increased peripheral pain with repeated lumbar extension.As described in a previous publication,  dynamic Midline sagittal plane imaging of the spine was performed using a receive-only surface coil and an ultrafast spoiled GRASS  pulse sequence. Images were obtained at a rate of 1 per second using the following parameters: repetition time (TR): 200 ms; echo time (TE): 18 ms; number of excitations: 1.0; matrix: 256 × 256; field of view (FOV): 28 × 21 cm; and a 7 mm section thickness with an interslice spacing of 1 mm.  The surfPrior to participation, informed consent was obtained as approved by the Institutional Review Boards of the University of Southern California and Stanford University. Two sets of images were acquired for each subject: 1) during the manual PA mobilization procedure and 2) during a PU maneuver. The order of testing was randomized for each subject.For the PA assessments, subjects were placed in the prone position on a sliding table within the MRI system, such that the spine and torso were within the opening between the vertical magnets Figure . For eacTo gain an insight into the posture of the lumbar spine in the starting prone position, we measured the angular position of the lumbar curvature with an inclinometer . To do so, we placed an inclinometer at the following anatomical locations S2, L3 and L1.After subjects were positioned within the MRI system, a series of sagittal plane "localizers" were obtained to ensure that the image plane captured the vertebral bodies and spinous processes of all lumbar vertebrae. Once the image plane was determined, continuous imaging (1 Hz) commenced.Following a static sagittal view of the lumbar spine in the resting position, a PA force was applied to the spinous processes at each vertebrae starting caudally at L5 and moving cranially to L1. The force was directed anteriorly in a direction perpendicular to the tangent of the arc of the lumbar lordosis. Sufficient force was applied to induce movement to the end of the available range of segmental motion and was comparable in magnitude to that of a "grade IV" as defined by Maitland.  The forcAll PA mobilizations were performed by a physical therapist with 15 years of manual therapy experience. A second investigator viewed the images on the MRI console to assure that the forces were being applied to the correct vertebral level. If an error was observed, imaging was repeated (this has occurred in three instances).For the PU assessment, subjects were placed in the prone position on the sliding table, such that the spine and torso were within the opening between the vertical magnets and the head and arms were outside of the MRI system Figure . For thiPrior to analysis, all images were transferred from the MRI system console to a Macintosh G3 computer . For purposes of this study, only the images at rest and at the end-range of segmental motion were analyzed. All data sets were again screened for quality of images and for pathology. Consequently, all images were deemed appropriate for further analysis. During screening, we noticed motion artifacts in the images where motion was expected. This helped in choosing the images for analysis. As the beginning of motion was readily observable, we chose the resting image for analysis at lest 5 images before motion was observed. The end of motion image was also chosen based clarity of the images, suggesting no motion. To assure ourselves that this assumption was correct, we have randomly selected 5 studies and have digitized three consecutive images deemed to be at the end of the range. This experiment assured us that our prior choice of the final image was correct.Lumbar segmental motion in the sagittal plane was quantified by measuring the intervertebral angle which was defined as the angle formed by lines delineating adjacent vertebral endplates Figure  27]. Se. Se27]. The same investigator, blinded to the group assignment, made all measurements using the National Institutes of Health (NIH) software . To establish the intra-tester reliability of angular measurements, dynamic MR images obtained from 5 healthy volunteers were measured on two separate occasions. Intraclass Correlation Coefficients (ICC) were found to be excellent, ranging from 0.95 to .99 for all subjects. The Standard Error of Measurement was determined to be between 0.4 and 0.66 degrees.For the PA analyses only the data from the motion-segment associated with the PA was used for the analysis. The data for both groups was screened for normality of distribution using Wilks-Shapiro W statistics. The symptomatic group did not exhibit normal distribution; therefore non-parametric statistics were used for further analyses.To determine the distribution of subjects outside of the normal range of segmental mobility, the norms were established based on the asymptomatic group's mean value ± 2 SD  demonstrated hyper-mobility at one motion segment during the PA assessment, while 3 subjects (15%) demonstrated hyper-mobility at one motion-segment during the PU maneuver. Individual lumbar motion-segment analysis revealed hyper-mobility in symptomatic subjects at L5 – S1  and L4 – L5   can be linked with recent disc injury, early disc degeneration or skeletal trauma. Subjects with skeletal trauma were excluded from this study. Hence relating intervertebral mobility to the condition of the disc is reasonable. As the mean age of the subjects in our study was in the early thirties, it is likely that their intervertebral discs were relatively normal, or at the very least, in the early stages of degeneration. The general trends in later life are associated with further disc degeneration and consequent joint degenerations leading to decreased motion (hypo-mobility). Therefore, the age of the subjects in this study may have had an influence on measured segmental mobility.The observation made from this study need to be viewed within its limitations. We relied on the experience of the operator  to apply sufficient about of force to produce end range at a motion-segment. We might have had introduced systematic bias into the PA experiment, by applying force from caudal to cranial, instead of randomizing the location of the force application. We studied a limited age range and are not able to compare between younger and older age groups.This study quantified in vivo segmental spinal mobility during a PA mobilization procedure and during a prone PU maneuver in persons with and without low back pain. The PA procedure identified hypermobilities at the L4-L5 and L5-S1 motion segments in the symptomatic group.The author(s) declare that they have no competing interests.KK, CMP, and RFL were responsible for the design of this study. CMP secured funding for this study. HW performed the data processing and analysis. MF and MG contributed to data collection and data interpretation. KB provided MRI expertise.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results.The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions.In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation. The complete sequence of the euchromatic region of the human genome provides a new opportunity to establish the complete catalogue of the human genes. Although automated gene prediction has improved greatly over the years and the human gene count is thought to be between 20,000 and 25,000 protein-coding genes , defininIn an effort to investigate and understand all the functional elements in the human genome, the ENCODE project (Encyclopedia of DNA Elements)  was estaThis annotation is used as a reference set by all the ENCODE consortium members. It also represents the standard to which the automated prediction programs were assessed during the ENCODE Genome Annotation Assessment Project (E-GASP) 05 workshop (see  in this The HAVANA group divides gene features into different categories of which only the first two (known and novel coding sequence (CDS)) are confidently predicted to be protein-coding genes. The common factor between all annotated gene structures is that they must be supported by transcriptional evidence, through homology to cDNA, expressed sequence tags (ESTs) and/or protein sequences. The following are the gene types first applied to the human chromosome 20 annotation  and lateKnown genes are identical to human cDNA or protein sequences and identified by a GeneID in Entrez Gene .Novel coding sequences have an open reading frame (ORF) and are identical, or have homology, to cDNAs or proteins but do not fall into the above category; these mRNA sequences are submitted to public databases, but they are not yet represented in Entrez Gene or have not yet received an official gene name from the nomenclature committee . They caNovel transcripts are as above but no ORF can be unambiguously assigned; these can be genuine non-coding genes or they may be partial protein-coding genes supported by limited evidence. They should be supported by at least three ESTs from independent sources (not originating from the same clone identifier).Putative genes are identical, or have homology, to spliced ESTs but lack a significant ORF and polyA features; these are generally short two or three exon genes or gene fragments.Pseudogenes (assumes no expressed evidence) have homology to proteins but generally suffer from a disrupted CDS and an active homologous gene can be found at another locus. This category can be further subdivided into processed or unprocessed pseudogenes. Sometimes these entries have an intact CDS or an open but truncated ORF, in which case there is other evidence used (for example genomic polyA stretches at the 3' end) to classify them as a pseudogene.Transcribed pseudogenes are not currently given a separate tag within GENCODE and are handled by creating a pseudogene object and an overlapping transcript object with the same locus name.To be experimentally confirmed (TEC) is used for non-spliced EST clusters that have polyA features. This category has been specifically created for the ENCODE project to highlight regions that could indicate the presence of novel protein coding genes that require experimental validation, either by 5' rapid amplification of cDNA ends (RACE) or RT-PCR to extend the transcripts or by confirming expression of the putatively encoded peptide with specific antibodies.Artefact gene is used to tag mistakes in the public databases (Ensembl/SwissProt/Trembl). Usually, these arise from high-throughput cDNA sequencing projects, which submit automatic annotation sometimes resulting in erroneous CDSs that are, for example, 3' untranslated regions (UTRs).The first release of the annotation of the 44 ENCODE regions was frozen on 29 April 2005 and was used in the E-GASP workshop. It contained 416 known loci, 26 novel CDS loci, 82 novel transcript loci, 78 putative loci, 104 processed pseudogenes and 66 unprocessed pseudogenes. The current version (release 02) was frozen on 14 October 2005. It contains 411 known loci, 30 novel CDS loci, 81 novel transcript loci, 83 putative loci, 104 processed pseudogenes and 66 unprocessed pseudogenes. The gene content has changed as a result of the experimental validation (see next section). In total, 2.9% of the nucleotides in the ENCODE regions (both strands considered separately) are covered by annotated exons (1.2% by coding and 1.7% by UTRs and non-coding), and 31% are transcribed (covered by annotated exons or introns).RNPC2  containing 2) gene has 37 variants, which is the highest number in the ENCODE regions, of which only 6 are annotated as coding.Multiple transcripts are annotated at any locus where supporting evidence is available. Thus, the 487 compiled GENCODE reference loci set  corresponds to 2,608 transcripts, of which 1,097 are coding. Of the coding loci (known and novel CDS), 78% have alternative splice forms (86% of the multi-exon gene loci), with an average of 5.7 variants per locus. Of the coding variants, approximately 70% have a complete CDS ; 54% of the coding loci have alternative CDS, indicating that diversity is lower at the protein level than at the transcript level as a substantial proportion of the alternative splice forms affect only the UTRs. The The initial HAVANA annotation was submitted for experimental verification Figure . First, Second, RT-PCR in 24 tissues was used for verifying transcript (novel and putative) structures by checking the splice junctions. All 360 splice junctions in the 161 novel and putative transcript loci were tested. Of those tested, 47 loci were validated, consisting of 31 novel and 15 putative transcripts. As expected, the success rate of RT-PCR was higher for the 'novel transcripts' (37%) than for the putative transcripts (19%). Bidirectional RACE was carried out for transcript loci with successfully validated splice junctions. This supported seven loci over their full length but did not extend them.Third, all annotated non-canonical sites  were tested by RT-PCR on 24 tissues. Of the annotated splice sites, 98% are canonical GT-AG and an additional 0.9% are GC-AG. There are 0.2% of AT-AC splice sites, most of them corresponding to canonical U12 introns . Other nFigure To examine whether the manual annotation had missed any coding loci, RT-PCR reactions in 24 tissues were also carried out for splice junctions from all those gene objects predicted by a panel of automated gene prediction algorithms before the E-GASP workshop , or has an entirely different CDS in a different frame. The latter often results in unusual structures, with multi-exon 5' and/or 3' UTRs that are at odds with rules governing re-initiation  and nonsGENCODE annotation uses only primary evidence; no predictions or RefSeq entries are used to support gene structures. This has the effect of reducing the risk of propagating any errors that may be present in the databases. The gene set annotated by GENCODE is supported using evidence from all available sources, human and non-human mRNAs, ESTs and proteins. The use of non-human evidence is supported by our analysis of four exons not present in our first pass annotation identified by the UNCOVER algorithm , two of The E-GASP workshop as part of the ENCODE project has highlighted the need for a high quality reference gene set that can be used to improve and validate prediction algorithms, as well as a scaffold for further experimentation. RT-PCR and 5' RACE of predicted exons outside the GENCODE annotation has currently not revealed additional multi-exon protein-coding genes. However, the experimental validation continually adds evidence for more splice variants. In addition, other technologies such as mapping RNA to tiling arrays , cap anaab initio predictions is run. The searches are run on a computer farm and stored in an Ensembl MySQL database using a modified Ensembl analysis pipeline system [ab initio prediction algorithms: Genescan [Before the process of manual annotation begins, an automated analysis pipeline for similarity searches and e system . All seae system ) are rune system  is used e system  to mask e system , and sige system . The Unie system  is searce system . The hide system  to proviGenescan  and FgenGenescan  for geneGenescan  to find Genescan , to predOnce the automated analysis is complete, the annotator uses a Perl/Tk based graphical interface, called 'otterlace', developed in-house to edit annotation data held in a separate MySQL database system . The int+ RNAs  using the BD SMART™ RACE cDNA amplification kit . Double-stranded cDNA synthesis and adaptor ligations to the synthesized cDNA were done according to the manufacturer's instructions. RACE fragments were separated on agarose gels and one or two strong single bands per gene purified and sequenced directly. Thus, successful RACE reactions appearing as a smear on the agarose gel would be discarded, therefore producing an approximate 54% success rate.Both 5' and 3' RACE were performed on 12 human poly(A)ab initio and four EST-based methods outside of HAVANA objects and 83 HAVANA novel and 78 putative transcripts (see Results and discussion for details).Similar amounts of 24 human cDNAs  were mixed with JumpStart REDTaq ReadyMix  and 4 ng/μl primers ) with a BioMek 2000 robot  as described and modified -45. The