Article Note: Maria Eugenia Morena-Barrio and Carlos Bravo-Perez contributed equally to this work. Javier Corral and Maria Luisa Lozano share senior authorship. Funding information This work was ...supported by the 19873/GERM/15 and 00001/COVI/20 projects (Fundacion Seneca) and by PI18/00598 (Instituto de Salud Carlos III & FEDER). ME M-B has a postdoctoral contract from Universidad de Murcia. C B-P is the recipient of a Rio Hortega contract for medical research investigator training from Instituto de Salud Carlos III (CM20/00094). B M-B has a predoctoral fellowship from Fundacion Seneca. R C has a predoctoral fellowship from Instituto de Salud Carlos III. CAPTION(S): Figure S1 Appendix S1 Byline: Maria Eugenia Morena-Barrio, Carlos Bravo-Perez, Belen Morena-Barrio, Christelle Orlando, Rosa Cifuentes, Jose Padilla, Antonia Minano, Sonia Herrero, Shally Marcellini, Nuria Revilla, Enrique Bernal, Jose Miguel Gomez-Verdu, Kristin Jochmans, Maria Teresa Herranz, Vicente Vicente, Javier Corral, Maria Luisa Lozano
Essentials
Vitamin K‐dependent coagulant factor deficiency (VKCFD) is a rare autosomal recessive disorder.
We describe a case of inherited VKCFD due to uniparental disomy.
The homozygous mutation ...caused the absence of GGCX isoform 1 and overexpression of Δ2GGCX.
Hepatic and non‐hepatic vitamin K‐dependent proteins must be assayed to monitor VKCFD treatment.
Summary
Background
Inherited deficiency of all vitamin K‐dependent coagulant factors (VKCFD) is a rare autosomal recessive disorder caused by mutations in the γ‐glutamyl carboxylase gene (GGCX) or the vitamin K epoxide reductase gene (VKORC1), with great heterogeneity in terms of both clinical presentation and response to treatment.
Objective
To characterize the molecular basis of VKCFD in a Spanish family.
Methods and Results
Sequencing of candidate genes, comparative genomic hybridization and massive sequencing identified a new mechanism causing VKCFD in the proband. Uniparental disomy (UPD) of chromosome 2 caused homozygosity of a mutation (c.44‐1G>A) resulting in aberrant GGCX splicing. This change contributed to absent expression of the mRNA coding for the full‐length protein, and to four‐fold overexpression of the smaller mRNA isoform lacking exon 2 (Δ2GGCX). Δ2GGCX might be responsible for two unexpected clinical observations in the patient: (i) increased plasma osteocalcin levels following vitamin K1 supplementation; and (ii) a mild non‐bleeding phenotype.
Conclusions
Our study identifies a new autosomal disease, VKCFD1, caused by UPD. These data suggest that the Δ2GGCX isoform may retain enzymatic activity, and strongly encourage the evaluation of both hepatic and non‐hepatic vitamin K‐dependent proteins to assess differing responses to vitamin K supplementation in VKCFD patients.
Reference values for Doppler parameters according to age and gender are recommended for the assessment of heart physiology, specifically for left ventricular (LV) diastolic function. In this study, ...we report normal reference ranges for Doppler parameters obtained in a large group of healthy volunteers. Echocardiographic data were acquired using state-of-the-art cardiac ultrasound equipment following Doppler acquisition and measurement protocols approved by the European Association of Cardiovascular Imaging.
A total of 449 (mean age: 45.8 ± 13.7 years) healthy volunteers (198 men and 251 women) were enrolled at the collaborating institutions of the Normal Reference Ranges for Echocardiography (NORRE) study. A comprehensive echocardiographic examination was obtained from all subjects following predefined protocols. The majority of the Doppler diastolic parameters (e', E/e') as well as right ventricle systolic s' wave velocity were similar in men and women. Left ventricle s' wave velocity was higher in men than in women. E wave and e' were higher in younger subjects and decreased progressively in the older ones. E/e' ratio increased with ageing. Septal e' <8 cm/s was present in 19.7% of the subjects in the 40-60 year group and in 55% of those in the ≥60 year group. However, the cut-off value of average E/e' or lateral E/e' remained <15 or 13, respectively, in the majority of patients.
The NORRE study provides the reference values for the most useful Doppler parameters in the evaluation of heart physiology. These data highlight the need of using age-specific reference values especially for the diagnosis of LV systolic and diastolic dysfunction and for the estimation of LV filling pressures.
Objectives
To study the relationship between coping strategies and prolonged grief disorder (PGD) in caregivers of patients with disorders of consciousness: vegetative state (VS) or minimally ...conscious state (MCS).
Materials and methods
Fifty‐three caregivers of 43 patients with VS or MCS were assessed using PG‐12 and Brief COPE‐28. Mean differences for each coping strategy between caregivers of patients with/without PGD were compared using Bonferroni‐adjusted t‐tests, and the size effect was calculated (Cohen′s d).
Results
The frequency of PGD was very high (n = 32; 60.40%). The most common coping strategies were problem‐focused: active coping (mean = 6.41; SD = 1.02), Instrumental support (mean = 6.41; SD = 1.06), Planning (mean = 6.32; SD = 1.01) and Acceptance (mean = 6.20; SD = 1.29). Acceptance predicted a lower presence of PGD (P = 0.001; Cohen′s d = 1.02), while Denial (P = 0.003; Cohen′s d = 0.98) and Self‐blame (P = 0.004, Cohen′s d = 0.91) increased the presence of PGD.
Conclusions
The caregivers of patients with VS or MCS show a high risk of PGD. Problem‐focused coping strategies are the most used. Acceptance is highly protective of PGD, and Denial and Self‐blame are associated with an increased presence of PGD. PGD in caregivers of patients in VS or MCS should be evaluated, Acceptance and problem‐focused strategies should be promoted, and Denial and Self‐blame should be diminished.
Cryo‐electron microscopy (cryoEM) has become a well established technique to elucidate the 3D structures of biological macromolecules. Projection images from thousands of macromolecules that are ...assumed to be structurally identical are combined into a single 3D map representing the Coulomb potential of the macromolecule under study. This article discusses possible caveats along the image‐processing path and how to avoid them to obtain a reliable 3D structure. Some of these problems are very well known in the community. These may be referred to as sample‐related (such as specimen denaturation at interfaces or non‐uniform projection geometry leading to underrepresented projection directions). The rest are related to the algorithms used. While some have been discussed in depth in the literature, such as the use of an incorrect initial volume, others have received much less attention. However, they are fundamental in any data‐analysis approach. Chiefly among them, instabilities in estimating many of the key parameters that are required for a correct 3D reconstruction that occur all along the processing workflow are referred to, which may significantly affect the reliability of the whole process. In the field, the term overfitting has been coined to refer to some particular kinds of artifacts. It is argued that overfitting is a statistical bias in key parameter‐estimation steps in the 3D reconstruction process, including intrinsic algorithmic bias. It is also shown that common tools (Fourier shell correlation) and strategies (gold standard) that are normally used to detect or prevent overfitting do not fully protect against it. Alternatively, it is proposed that detecting the bias that leads to overfitting is much easier when addressed at the level of parameter estimation, rather than detecting it once the particle images have been combined into a 3D map. Comparing the results from multiple algorithms (or at least, independent executions of the same algorithm) can detect parameter bias. These multiple executions could then be averaged to give a lower variance estimate of the underlying parameters.
Single‐particle analysis (SPA) by cryo‐electron microscopy comprises the estimation of many parameters along its image‐processing pipeline. Overfitting observed in SPA is normally due to misestimated parameters, and the only way to identify these is by comparing the estimates of multiple algorithms or, at least, multiple executions of the same algorithm.
Introduction
Factor XI (FXI) deficiency is a rare disorder with molecular heterogeneity in Caucasians but relatively frequent and molecularly homogeneous in certain populations.
Aim
To characterize ...FXI deficiency in a Spanish town of 60 000 inhabitants.
Methods
A total of 324 764 APTT tests were screened during 20 years. FXI was evaluated by FXI:C and by Western blot. Genetic analysis of F11 was performed by sequencing, multiplex ligation‐dependent probe amplification and genotyping.
Results
Our study identified 46 unrelated cases and 170 relatives with FXI deficiency carrying 12 different genetic defects. p.Cys56Arg, described as founder mutation in the French‐Basque population, was identified in 109 subjects from 24 unrelated families. This mutation was also identified in 2% of the general population. p.Cys416Tyr, c.1693G>A and p.Pro538Leu were identified in 7, 6 and 2 unrelated families, respectively. NGS analysis of the whole F11 gene revealed a common haplotype for each of the four recurrent mutations, suggesting a founder effect. The analysis of plasma FXI of four p.Pro538Leu homozygous carriers revealed that this variant was not activated by FXIIa. We identified four mutations previously described in other Caucasian subjects with FXI deficiency (p.Lys536Asn; p.Thr322Ile, p.Arg268Cys and c.325G>A) and four new gene defects: p.(Cys599Tyr) potentially causing a functional deficiency, p.(Ile426Thr), p.(Ile592Thr) and the first worldwide duplication of 1653 bp involving exons 8 and 9. Bleeding was rare and mild.
Conclusions
Our population‐cohort study supplies new evidences that FXI deficiency in Caucasians is more common than previously thought and confirmed the wide underlying genetic heterogeneity, caused by both recurrent and sporadic mutations.
Display omitted
•Interoperability among all integrated software (with some limitations explicitly mentioned in the paper).•The whole cryoET data processing pipeline is covered.•Modern visualization ...tools for cryoET data, like segmentation, oriented 3D coordinates, or a tilt series CTF analyzer.•Manages all the metadata and image file conversions.•Handles incompatibilities between different software packages.•Prevents users from scripting, losing track of the completed processing attempts, and using a logbook.
Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline.
The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project.
The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.
Display omitted
The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from ...yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achieved.
PDF
