Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of α-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual α-dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with Walker-Warburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This finding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modification of the α-dystroglycan protein

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Measurement of promoter band intensities on chromatin blot confirmed mating-type dependent changes.

<p>Measurements were made on the autoradiograph of the chromatin blot of strains with a telomeric reporter at XI-L. Intensities were measured along a vertical line drawn through the three promoter bands produced by digestion with the highest concentration of MNase I. The graphs show intensity versus distance in inches along the line. Arrows indicate the band positions. The left hand peak corresponds to the top promoter band on the auroradiograph. Strains on graphs: haploid (blue), dipoid (red), diploid <i>mata-</i>delta (green).</p

Levels of protein from <i>hml::YFP</i> reporter increase with cell size.

<p>Diploid strains with a range of cell size were created by mating appropriate strains from the haploid Yeast Deletion Collection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039044#pone.0039044-Winzeler1" target="_blank">[19]</a> to the haploid strain containing the <i>hml::YFP</i> reporter. YFP levels were measured by flow cytometry in these strains (pink squares) and in haploid (red diamond), diploid (blue triangle) and triploid (black circle) strains. Mode fluorescence was plotted versus cell volume in µm³ (measured by microscopy).</p

Level of protein from <i>hml::YFP</i> depends on ploidy but not mating-type.

<p>Yeast strains containing a single <i>YFP</i> reporter at <i>hml</i> had fluorescence levels measured by flow cytometry. (A) Strains on histogram plot: haploid (n, red), diploid (2n, blue), diploid with one <i>MAT</i> deleted (2n*, green) and haploid made heterozygous for <i>MAT</i> (n’, purple). (B) Strains on histogram plot: haploid (n, red), diploid (2n, blue), triploid (3n, orange).</p

Neither Sir3 nor Sir4 are limiting at <i>HML</i> in diploid cells.

<p>A single copy of <i>SIR3</i> or <i>SIR4</i> was deleted from diploid cells and fluorescence levels were measured by flow cytometry. Strains on histogram plot: haploid (n, red), diploid (2n, blue), <i>SIR3</i>/<i>sir3-</i>Δ (pink), <i>SIR4</i>/<i>sir4-</i>Δ (turquoise).</p

Level of reporter fluorescence measured by flow cytometry and RNA level as ratio to that in haploid cells.

*<p>modal value of fluorescence. wt: wild-type.</p

Transcriptional silencing at telomeres does not change with ploidy.

<p>mRNA levels were measured by QRT-PCR and compared to the levels in haploid cells. The values plotted are ratios to the amounts in the haploid cells. They are mean values from 3 separate RT-PCR reactions. The error bars show 2 standard deviations. The strains had the reporter positioned at XI-L (n XI L, 2n XI L, 2n* XI L, n <i>sir3</i>Δ), at <i>URA3</i> native locus (n native), or at III-R (n III R).</p

Mating-type dependent changes at the promoter of the <i>URA3-yEGFP</i> telomeric reporter.

<p>Southern blots of micrococcal nuclease I digests of chromatin from haploid (n), diploid (2n) and diploid <i>mata</i>-Δ (2n*) cells with <i>URA3-yEGFP</i> reporter at telomere XI-L. Position of <i>URA3-yEGFP</i> reporter is shown: promoter as grey box with TATA box (T) and ORF as blue box. Centromeric to the reporter are the regularly spaced hypersensitive sites typical of heterochromatin (blue arrows) and around the promoter are three hypersensitive sites whose relative intensity varies (black arrows). Control MNase I digests of deproteinized DNA (N), marker bands generated by digestion with <i>Stu</i>I and <i>Pst</i>I (M), and sizes on blot (kb) are also shown.</p

Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in saccharomyces cerevisiae

The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM) loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA) media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L), changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae