Single Cell Transcriptomics Reveal Abnormalities in Neurosensory Patterning of the Chd7 Mutant Mouse Ear

The chromatin remodeling protein CHD7 is critical for proper formation of the mammalian inner ear. Humans with heterozygous pathogenic variants in CHD7 exhibit CHARGE syndrome, characterized by hearing loss and inner ear dysplasia, including abnormalities of the semicircular canals and Mondini malformations. Chd7Gt/+ heterozygous null mutant mice also exhibit dysplastic semicircular canals and hearing loss. Prior studies have demonstrated that reduced Chd7 dosage in the ear disrupts expression of genes involved in morphogenesis and neurogenesis, yet the relationships between these changes in gene expression and otic patterning are not well understood. Here, we sought to define roles for CHD7 in global regulation of gene expression and patterning in the developing mouse ear. Using single-cell multiplex qRT-PCR, we analyzed expression of 192 genes in FAC sorted cells from Pax2Cre;mT/mGFP wild type and Chd7Gt/+ mutant microdissected mouse otocysts. We found that Chd7 haploinsufficient otocysts exhibit a relative enrichment of cells adopting a neuroblast (vs. otic) transcriptional identity compared with wild type. Additionally, we uncovered disruptions in pro-sensory and pro-neurogenic gene expression with Chd7 loss, including genes encoding proteins that function in Notch signaling. Our results suggest that Chd7 is required for early cell fate decisions in the developing ear that involve highly specific aspects of otic patterning and differentiation

Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a “second hit,” that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome

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

SHP2 regulates chondrocyte terminal differentiation, growth plate architecture and skeletal cell fates.

Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC) patients causes benign cartilage tumors on the bone surface (exostoses) and within bones (enchondromas). To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the data are consistent with paracrine signaling from SHP2-deficient cells causing SHP2-sufficient cells to be incorporated into the lesions

MEK1/2 inhibition or SHP2 depletion alters the expression of transcription factors with known roles in chondrocyte maturation.

<p>Schematic diagrams of genes encoding transcription factors (solid boxes) or their known target genes (empty boxes) that significantly decreased in abundance (blue) or increased in abundance (red) in U0126-treated pellets at w3. All transcription factors also showed similar increases or decreases in <i>Ptpn11</i> cKO pellets at w3, but not all reached statistical significance. A: Transcription factors with known roles in promoting chondrocyte hypertrophy (upper green arrow), or in promoting the terminal differentiation of chondrocytes and/or their replacement by bone (lower green arrow). B: Transcription factors whose known target genes also significantly changed in abundance in U0126-treated pellets. Arrows indicate transcriptional activation while bar-headed lines indicate transcriptional repression.</p

Disordered maturation zones and delayed terminal differentiation in mouse vertebral growth plates following mosaic postnatal inactivation of <i>Ptpn11</i> in chondrocytes.

<p>Immunohistochemistry performed on tissue sections from the vertebral growth plates of Ctrl mice (A,D) or the growth plates (B,E) or enchondroma-like lesions (C) in Col2a1-cKO mice. A: Ctrl growth plate showing COLX, p-ERK1/2 and FRA1 immunoreactivity in the hypertrophic zone. COLX immunoreactivity is also observed in the calcifying region of the annulus fibrosus that lies directly above the growth plate. B: Col2a1-cKO growth plate showing ectopic COLX-positive clusters at the top of the growth plate (a) and ectopic COLX-negative clusters at the bottom of the growth plate (b). Furthermore, hypertrophic chondrocytes within the center of the growth plate display disorganized, scattered p-ERK1/2 and FRA1 immunoreactivity (c). C: Enchondroma-like lesions contain both COLX-positive and COLX-negative chondrocytes. D: Ctrl growth plate showing SPP1 immunoreactivity at the bottom of the hypertrophic zone and in mineralizing annulus fibrosus cells. E: In Col2a1-cKO growth plates, expanded regions of SPP1-negative hypertrophic chondrocytes are observed (red arrows) and only a few scattered hypertrophic chondrocytes are SPP1-positive (black arrows). IHC tissue sections were counterstained with hematoxylin. Scale bars  =  25 µm.</p

Lineage tracing of SHP2-depleted chondrocytes in mouse vertebral growth plates following mosaic postnatal <i>Ptpn11</i> inactivation.

<p>Tissue sections of lumbar vertebral growth plates (GP) and intervertebral disks (IVD) from <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl</i>^/<i>fl</i> (Col2a1-cKO) or <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl/+</i> (Ctrl) mice that had been administered tamoxifen for 5 days, starting at w1, and sacrificed at w7. A-C: Tissue sections stained with Alcian Blue and Nuclear Fast Red. In Col2a1-cKO mice, the vertebral growth plates are enlarged and disorganized, with ectopic areas of ossification inside the growth plate (red arrow in B) and enchondroma-like lesions below the growth plate (C). D-G: Merged fluorescent images showing cells in which the <i>mTmG</i> reporter allele has (green fluorescence) or has not (red fluorescence) been recombined by Cre recombinase. Note that approximately 50% of growth plate chondrocytes fluoresce green, and in Col2a1-cKO mice, the expanded regions of the growth plate (E), lateral outgrowths (F) and enchondroma-like lesions (G) contain both green and red fluorescing cells. Dashed-white lines outline the cartilage. Scale bars  =  100 µm.</p

Identifying transcripts that change in abundance following SHP2 depletion or MEK1/2 inhibition in pellet cultures.

<p>A: Immunodetection of SHP2, phospho-ERK1/2, total ERK1/2, and phospho-AKT in chondrocyte pellet cultures. Top: pellets from <i>Ptpn11^fl/–</i> or <i>Tg</i>(<i>CMV-CreERT2); Ptpn11^fl/–</i> mice were treated daily with 4-OHT or vehicle control (EtOH, -) and harvested after 1 and 3 weeks. Bottom: pellets from wild-type mice were treated daily with a MEK1/2 inhibitor (U0126), or vehicle control (DMSO, -), and harvested after 1 and 3 weeks. B: Number of transcripts that had significantly lower (blue) or higher (red) abundance in treated pellets compared to vehicle treated pellets, either at w1 or w3. Transcripts that changed in abundance in <i>Ptpn11</i> cKO pellets were excluded if they exhibited similar changes in wild-type pellets treated with 4-OHT.</p