Sequence Features and Transcriptional Stalling within Centromere DNA Promote Establishment of CENP-A Chromatin

Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on ∼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription-TFIIS and Ubp3-assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an environment of pervasive low quality RNAPII transcription that is an important determinant of CENP-ACnp1 assembly. These observations emphasise roles for both genetic and epigenetic processes in centromere establishment

Association of HLA-DRB1 amino acid residues with giant cell arteritis: genetic association study, meta-analysis and geo-epidemiological investigation

Introduction: Giant cell arteritis (GCA) is an autoimmune disease commonest in Northern Europe and Scandinavia. Previous studies report various associations with HLA-DRB1*04 and HLA-DRB1*01; HLA-DRB1 alleles show a gradient in population prevalence within Europe. Our aims were (1) to determine which amino acid residues within HLA-DRB1 best explained HLA-DRB1 allele susceptibility and protective effects in GCA, seen in UK data combined in meta-analysis with previously published data, and (2) to determine whether the incidence of GCA in different countries is associated with the population prevalence of the HLA-DRB1 alleles that we identified in our meta-analysis. Methods: GCA patients from the UK GCA Consortium were genotyped by using single-strand oligonucleotide polymerization, allele-specific polymerase chain reaction, and direct sequencing. Meta-analysis was used to compare and combine our results with published data, and public databases were used to identify amino acid residues that may explain observed susceptibility/protective effects. Finally, we determined the relationship of HLA-DRB1*04 population carrier frequency and latitude to GCA incidence reported in different countries. Results: In our UK data (225 cases and 1378 controls), HLA-DRB1*04 carriage was associated with GCA susceptibility (odds ratio (OR) = 2.69, P = 1.5×10 −11 ), but HLA-DRB1*01 was protective (adjusted OR = 0.55, P = 0.0046). In meta-analysis combined with 14 published studies (an additional 691 cases and 4038 controls), protective effects were seen from HLA-DR2, which comprises HLA-DRB1*15 and HLA-DRB1*16 (OR = 0.65, P = 8.2×10 −6 ) and possibly from HLA-DRB1*01 (OR = 0.73, P = 0.037). GCA incidence (n = 17 countries) was associated with population HLA-DRB1*04 allele frequency (P = 0.008; adjusted R 2 = 0.51 on univariable analysis, adjusted R 2 = 0.62 after also including latitude); latitude also made an independent contribution. Conclusions: We confirm that HLA-DRB1*04 is a GCA susceptibility allele. The susceptibility data are best explained by amino acid risk residues V, H, and H at positions 11, 13, and 33, contrary to previous suggestions of amino acids in the second hypervariable region. Worldwide, GCA incidence was independently associated both with population frequency of HLA-DRB1*04 and with latitude itself. We conclude that variation in population HLA-DRB1*04 frequency may partly explain variations in GCA incidence and that HLA-DRB1*04 may warrant investigation as a potential prognostic or predictive biomarker

Technical Note: Intensity-based quality assurance criteria for deformable image registration in image-guided radiotherapy

International audienceBackground: Deformable image registration is increasingly used in radiotherapy to adapt the treatment plan and accumulate the delivered dose. Consequently, clinical workflows using deformable image registration require quick and reliable quality assurance to accept registrations. Additionally, for online adaptive radiotherapy, quality assurance without the need for an operator to delineate contours while the patient is on the treatment table is needed. Established quality assurance criteria such as the Dice similarity coefficient or Hausdorff distance lack these qualities and also display a limited sensitivity to registration errors beyond soft tissue boundaries.Purpose: The purpose of this study is to investigate the existing intensity-based quality assurance criteria structural similarity and normalized mutual information for their ability to quickly and reliably identify registration errors for (online) adaptive radiotherapy and compare them to contour-based quality assurance criteria.Methods: All criteria were tested using synthetic and simulated biomechanical deformations of 3D MR images as well as manually annotated 4D CT data. The quality assurance criteria were scored for classification performance, for their ability to predict the registration error, and for their spatial correlation with the registration error.Results: We found that besides being fast and operator-independent, the intensitybased criteria have the highest area under the receiver operating characteristic curve and provide the best input for models to predict the registration error on all data sets. Structural similarity furthermore provides spatial information with a higher spatial correlation to the benchmark than the inverse consistency error, Jacobian determinant, and curl magnitude.Conclusions: Intensity-based quality assurance criteria can provide the required confidence in decisions about using mono-modal registrations in clinical workflows. They thereby enable automated quality assurance for deformable image registration in adaptive radiotherapy treatments

Quantitative investigation of dose accumulation errors from intra-fraction motion in MRgRT for prostate cancer

International audienceAccurate spatial dose delivery in radiotherapy is frequently complicated due to changes in the patient's internal anatomy during and in-between therapy segments. The recent introduction of hybrid MRI radiotherapy systems allows unequaled soft-tissue visualization during radiation delivery and can be used for dose reconstruction to quantify the impact of motion. To this end, knowledge of anatomical deformations obtained from continuous monitoring during treatment has to be combined with information on the spatio-temporal dose delivery to perform motion-compensated dose accumulation (MCDA). Here, the influence of the choice of deformable image registration algorithm, dose warping strategy, and MR image resolution and SNR on the resulting MCDA is investigated. For a quantitative investigation, four 4D MRI-datasets representing typical patient observed motion patterns are generated using finite element modeling and serve as a gold standard. Energy delivery is simulated intra-fractionally in the deformed image space and, subsequently, MCDA-processed. Finally, the results are substantiated by comparing MCDA strategies on clinically acquired patient data. It is shown that MCDA is needed for correct quantitative dose reconstruction. For prostate treatments, using the energy per mass transfer dose warping strategy has the largest influence on decreasing dose estimation errors

Integration of operator-validated contours in deformable image registration for dose accumulation in radiotherapy

Background and Purpose: Deformable image registration (DIR) is a core element of adaptive radiotherapy workflows, integrating daily contour propagation and/or dose accumulation in their design. Propagated contours are usually manually validated and may be edited, thereby locally invalidating the registration result. This means the registration cannot be used for dose accumulation. In this study we proposed and evaluated a novel multi-modal DIR algorithm that incorporated contour information to guide the registration. This integrates operator-validated contours with the estimated deformation vector field and warped dose. Materials and Methods: The proposed algorithm consisted of both a normalized gradient field-based data-fidelity term on the images and an optical flow data-fidelity term on the contours. The Helmholtz-Hodge decomposition was incorporated to ensure anatomically plausible deformations. The algorithm was validated for same- and cross-contrast Magnetic Resonance (MR) image registrations, Computed Tomography (CT) registrations, and CT-to-MR registrations for different anatomies, all based on challenging clinical situations. The contour-correspondence, anatomical fidelity, registration error, and dose warping error were evaluated. Results: The proposed contour-guided algorithm considerably and significantly increased contour overlap, decreasing the mean distance to agreement by a factor of 1.3 to 13.7, compared to the best algorithm without contour-guidance. Importantly, the registration error and dose warping error decreased significantly, by a factor of 1.2 to 2.0. Conclusions: Our contour-guided algorithm ensured that the deformation vector field and warped quantitative information were consistent with the operator-validated contours. This provides a feasible semi-automatic strategy for spatially correct warping of quantitative information even in difficult and artefacted cases

Epidural analgesia at trial of labor after cesarean (TOLAC): a significant adjunct to successful vaginal birth after cesarean (VBAC)

Epidural analgesia has been considered a risk factor for labor dystocia at trial of labor after cesarean (TOLAC) and uterine rupture. We evaluated the association between exposure to epidural during TOLAC and mode of delivery and maternal-neonatal outcomes.A single center retrospective study of women that consented to TOLAC within a strict protocol between 2006 and 2013. Epidural "users" were compared to "non-users". Primary outcome was the mode of delivery: repeat in-labor cesarean or vaginal birth after cesarean (VBAC). Secondary outcomes were maternal/neonatal morbidities. Univariate/multivariate analyses for associations between epidural and mode of delivery were adjusted for significant covariates/mediators.Of a total of 105,471 births registered, 9464 (9.0%) were eligible for TOLAC; 7149 (75.5%) women consented to TOLAC, among which 4081 (57.1%) had epidural analgesia. The in labor cesarean rate was significantly lower for the epidural "users" 8.7% vs. "non-users" 11.8%, P<0.0001, with a parallel increased rate of instrumental delivery. Uterine rupture rates were comparable: 0.4% and 0.29%, respectively (P=0.31). The adjusted multivariate model showed that epidural "users" were more likely to experience a VBAC, odds ratio (OR) 4.58 [3.67; 5.70]; P<0.0001 with a similar rate of adverse maternal-neonatal outcomes.Epidural analgesia at TOLAC may emerge as a safe and significant adjunct for VBAC

Epidural analgesia at trial of labor after cesarean (TOLAC): a significant adjunct to successful vaginal birth after cesarean (VBAC)

Epidural analgesia has been considered a risk factor for labor dystocia at trial of labor after cesarean (TOLAC) and uterine rupture. We evaluated the association between exposure to epidural during TOLAC and mode of delivery and maternal-neonatal outcomes.A single center retrospective study of women that consented to TOLAC within a strict protocol between 2006 and 2013. Epidural "users" were compared to "non-users". Primary outcome was the mode of delivery: repeat in-labor cesarean or vaginal birth after cesarean (VBAC). Secondary outcomes were maternal/neonatal morbidities. Univariate/multivariate analyses for associations between epidural and mode of delivery were adjusted for significant covariates/mediators.Of a total of 105,471 births registered, 9464 (9.0%) were eligible for TOLAC; 7149 (75.5%) women consented to TOLAC, among which 4081 (57.1%) had epidural analgesia. The in labor cesarean rate was significantly lower for the epidural "users" 8.7% vs. "non-users" 11.8%, P&lt;0.0001, with a parallel increased rate of instrumental delivery. Uterine rupture rates were comparable: 0.4% and 0.29%, respectively (P=0.31). The adjusted multivariate model showed that epidural "users" were more likely to experience a VBAC, odds ratio (OR) 4.58 [3.67; 5.70]; P&lt;0.0001 with a similar rate of adverse maternal-neonatal outcomes.Epidural analgesia at TOLAC may emerge as a safe and significant adjunct for VBAC