Facioscapulohumeral Dystrophy: Incomplete Suppression of a Retrotransposed Gene

Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

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

Immunodetection of Human Double Homeobox 4

Double homeobox 4 (DUX4) is a candidate disease gene for facioscapulohumeral dystrophy (FSHD), one of the most common muscular dystrophies characterized by progressive skeletal muscle degeneration. Despite great strides in understanding precise genetics of FSHD, the molecular pathophysiology of the disease remains unclear. One of the major limitations has been the availability of appropriate molecular tools to study DUX4 protein. In the present study, we report the development of five new monoclonal antibodies targeted against the N- and C-termini of human DUX4, and characterize their reactivity using Western blot and immunofluorescence staining. Additionally, we show that expression of the canonical full coding DUX4 induces cell death in human primary muscle cells, whereas the expression of a shorter splice form of DUX4 results in no such toxicity. Immunostaining with these new antibodies reveals a differential effect of two DUX4 isoforms on human muscle cells. These antibodies will provide an excellent tool for investigating the role of DUX4 in FSHD pathogenesis

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis

Abstract Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor β (TGFβ) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFβ signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFβ family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings reveal dysfunction of BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals

Highly enriched repeat types among DUX4 binding sites.

1<p>We show only repeat types with ≥2-fold enrichment among either the ChIP-seq peaks (showing only those with ≥100 peaks) or among the ChIP-seq reads (showing only those with ≥1000 reads). Repeat types are shown sorted by read-based enrichment estimate. Full results for all repeat types are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen.1003947.s015" target="_blank">Table S2</a>, and are given aggregated by repeat family in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen.1003947.s016" target="_blank">Table S3</a>.</p

Examples of DUX4-bound repeats that function as alternative promoters for lncRNAs or antisense transcripts.

<p>In each panel, thin red boxes depict DUX4-bound repetitive elements. As in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen-1003947-g004" target="_blank">Figure 4</a>, exons of previously annotated transcripts are depicted as green boxes, with alternative DUX4-induced exons as empty boxes (not to scale). Arrows show the direction of transcription. Diagonal lines show splicing, with alternative splice forms shown above and below the exons. (A) A lncRNA initiated at an MLT1C element. Two exons of the DUX4-activated lncRNA overlap with exons of previously described lncRNAs TCONS_00003193, TCONS_00002742, TCONS_00002660 and TCONS_00003194 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen.1003947-Cabili1" target="_blank">[42]</a>. (B) A lncRNA initiated at an THE1C element that shares the second exon of lncRNA TCONS_00022347 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen.1003947-Cabili1" target="_blank">[42]</a>. Additional lncRNAs on the opposite strand also initiate in this region - for clarity, only one is depicted here. (C) An antisense RNA initiated at a DUX4-bound MLT1D element that overlaps the first exon of <i>DDX10</i>.</p

Many repeat types are enriched among DUX4 binding sites.

<p>(A) ∼2/3 of DUX4 binding-sites are in repetitive elements, compared to ∼45% of the human genome. (B) Comparing repeat family proportions among DUX4 binding-sites with genome-wide fractions shows ∼10-fold MaLR enrichment. (C) A peak-based method of estimating repeat enrichment uses uniquely-mapped reads, so is blind to recently active repeats; however, it ignores background reads so provides a more sensitive enrichment measure than the read-based estimate (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen-1003947-g001" target="_blank">Figures 1D, 1E</a>). 32 repeat types (red) are enriched ≥2-fold with ≥100 peaks (arbitrary thresholds) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen-1003947-t001" target="_blank">Tables 1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen.1003947.s015" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen.1003947.s016" target="_blank">S3</a>); 21 (orange) are rarer in the genome (10–99 peaks) but enriched ≥4-fold. The log10-scaled x-axis shows the proportion of peaks expected to overlap each repeat type if DUX4 binding sites had uniform genomic distribution; the log10-scaled y-axis shows observed proportions. The dashed line represents no enrichment. In all panels, “+” symbols represent MaLR elements and “x” datapoints represent repeat types for which no peaks/reads were observed – these are given an arbitrary low (non-zero) value to ensure visibility on log-scaled plots. (D) The read-based enrichment estimation method examines highly similar repeats as well as uniquely-mappable sequences, but gives a “dampened” enrichment measure due to background reads in ChIP-seq samples (see Methods). 25 repeat types (dark blue) are enriched ≥2-fold, with ≥1000 reads (arbitrary thresholds); 3 (light blue) are rarer among ChIP-seq reads (100–999 reads) but enriched ≥4-fold. (E) The peak-based (x-axis) and read-based (y-axis) methods yield similar results. 19 repeat types (green datapoints, thresholds as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003947#pgen-1003947-g001" target="_blank">Figures 1A and B</a> for red and dark blue points) were enriched in both analyses; 13 enriched only by the peak-based method (red), and 6 enriched only by the read-based method (blue). Additional repeats (gray datapoints, upper-right quadrant) appear enriched by both methods, but are rare in the genome so do not exceed our arbitrary peak/read thresholds.</p