A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Disability Testing and Retirement

<p>Images were extracted from the movie presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097148#pone.0097148.s020" target="_blank">movie S12</a>. The time interval after the starting point of the movie is indicated in hours (h). Images were extracted from three channels (bright field, BF, left column; GFP, middle column; GFP and mCherry, GFP-Che, right column). The dynamics of the localization of the GFP fusions was followed in <i>Msm</i>::<i>mCherry-wag31</i> bacteria expressing the MmpL3-GFP fusions. The presence of MmpL3 in septa is indicated by white arrows in the right column. The “croissant”-shaped accumulation of MmpL3 at the poles is indicated by white arrows in the middle column. The position of mCherry-Wag31 at the poles and septa is clearly visible. Magnification: 630 X. Scale bar: 5 µm).</p

Polar colocalization of FAS-II proteins with Wag31.

<p>(A) Enlarged images of wide-field microscopy experiments on GFP-fusion and mCherry-Wag31 fusion-expressing bacteria. GFP fluorescence images (GFP; leftmost column) together with mCherry fluorescence images (Che, middle column) of <i>Msm::mCherry-wag31</i> expressing GFP fusions with MabA, InhA, KasA, KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar colocalization of the fusions. Magnification and scale bars are identical to those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097148#pone-0097148-g002" target="_blank">Figure? 2A</a>. (B) Bar graph representation of GFP polar indices of <i>Msm::mCherry-wag31</i> strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as mean ± SD. Data were processes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097148#pone-0097148-g002" target="_blank">Figure? 2B</a>. Statistical <i>t</i>-tests were performed to determine the differences between the polar indices of GFP-InhA (dark grey bar) and GFP-KasA (light grey bar) and of GFP-KasA and GFP-KasB (white bar). The <i>p</i> values of indicated unpaired t-tests are symbolized by asterisks (*, <i>p</i> = 0.0271 for InhA-KasA), (*, <i>p</i> = 0.0186 for KasA-KasB). (C) Bar graph representation of GFP-mCherry colocalization indices of <i>Msm::mCherry-wag31</i> strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as means ± SD. Data were processed as in panel B; no significant differences were found between colocalization indices. (D) Analysis of maximal mCherry-Wag31 (Che-Wag31, left column) and GFP-FAS-II fusion (GFP fusion, right column) fluorescence position within individual <i>Msm::mCherry-wag31</i> bacteria expressing each GFP fusion after 6 hours of induction. Each type of bacterium was scanned on both channels. Each dot represents the position of maximum fluorescence of the scans expressed in % of the highest values. Each dot was plotted against its position in the bacterium with the length of bacteria reported to 1. The names of the FAS-II proteins fused with GFP are indicated. Ø refers to the <i>Msm::mCherry-wag31</i> strain containing GFP alone.</p

Dynamics of GFP-MabA localization.

<p>Images were extracted from the movie presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097148#pone.0097148.s010" target="_blank">movie S2</a>. The time interval after the starting point of the movie is indicated in hour (h). Merge bright field and GFP channel images (BF-GFP, left column) and GFP channel images (GFP, right column) were extracted in order to follow the dynamics of representative GFP-MabA foci. Two bacteria and their daughter cells were schematized (far right). Representative foci (in green) are labeled (a to f) and followed, when possible, in the daughter cells. Superscript numbering represents potential foci splitting. White arrows indicate the localization of the main foci on GFP images. Magnification and scale are identical to those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097148#pone-0097148-g002" target="_blank">Figure 2A</a>.</p

<i>Mycobacterium tuberculosis</i> Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum

<div><p>Understanding the mechanism that controls space-time coordination of elongation and division of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential <i>in vivo</i>. Using a dynamic approach, we localized <i>Mtb</i> enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of <i>Mtb</i>: <i>M. smegmatis</i>. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.</p></div

Schematic representation of mycobacterial polar growth and establishment of bacterial poles.

<p>Two division cycles (D1 and D2) are presented. The active biosynthesis of lateral peptidoglycan at the poles and biosynthesis of septal peptidoglycan at the future pole (septal poles) are symbolized with curved arrows. The old pole (in red) and the new pole (in blue); are both represented and refer to the first division event (D1).</p

Dynamic of GFP-MabA/Wag31 colocalization.

<p>Images were extracted from the movie presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097148#pone.0097148.s015" target="_blank">movie S7</a>. The time interval after the starting point of the movie is indicated in hours (h). Images were extracted from three channels (bright field, BF, left column; GFP, middle column; mCherry, Che, right column) in order to follow the dynamics of representative GFP-MabA and mCherry-Wag31 foci. Two bacteria, numbered 1 and 2, and their daughter cells, identified by superscript numbering are schematized on the right. Magnification: 630 X. Scale bar: 5 µm.</p

Development and Validation of a New Risk Prediction Score for Life-Threatening Ventricular Tachyarrhythmias in Laminopathies

International audienceBackground - An accurate estimation of the risk of life-threatening (LT) ventricular tachyarrhythmia (VTA) in patients with LMNA mutations is crucial to select candidates for implantable cardioverter-defibrillator implantation. Methods - We included 839 adult patients with LMNA mutations, including 660 from a French nationwide registry in the development sample, and 179 from other countries, referred to 5 tertiary centers for cardiomyopathies, in the validation sample. LTVTA was defined as (1) sudden cardiac death or (2) implantable cardioverter defibrillator-treated or hemodynamically unstable VTA. The prognostic model was derived using the Fine-Gray regression model. The net reclassification was compared with current clinical practice guidelines. The results are presented as means (SD) or medians [interquartile range]. Results - We included 444 patients, 40.6 (14.1) years of age, in the derivation sample and 145 patients, 38.2 (15.0) years, in the validation sample, of whom 86 (19.3%) and 34 (23.4%) experienced LTVTA over 3.6 [1.0-7.2] and 5.1 [2.0-9.3] years of follow-up, respectively. Predictors of LTVTA in the derivation sample were: male sex, nonmissense LMNA mutation, first degree and higher atrioventricular block, nonsustained ventricular tachycardia, and left ventricular ejection fraction (https://lmna-risk-vta.fr). In the derivation sample, C-index (95% CI) of the model was 0.776 (0.711-0.842), and the calibration slope 0.827. In the external validation sample, the C-index was 0.800 (0.642-0.959), and the calibration slope was 1.082 (95% CI, 0.643-1.522). A 5-year estimated risk threshold ≥7% predicted 96.2% of LTVTA and net reclassified 28.8% of patients with LTVTA in comparison with the guidelines-based approach. Conclusions - In comparison with the current standard of care, this risk prediction model for LTVTA in laminopathies significantly facilitated the choice of candidates for implantable cardioverter defibrillators. Clinical trial registration - URL: https://www.clinicaltrials.gov. Unique identifier: NCT03058185