Craniofacial dysmorphology in Down syndrome is caused by increased dosage of Dyrk1a and at least three other genes

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), occurs in 1 in 800 live births and is the most common human aneuploidy. DS results in multiple phenotypes, including craniofacial dysmorphology, which is characterised by midfacial hypoplasia, brachycephaly and micrognathia. The genetic and developmental causes of this are poorly understood. Using morphometric analysis of the Dp1Tyb mouse model of DS and an associated mouse genetic mapping panel, we demonstrate that four Hsa21-orthologous regions of mouse chromosome 16 contain dosage-sensitive genes that cause the DS craniofacial phenotype, and identify one of these causative genes as Dyrk1a. We show that the earliest and most severe defects in Dp1Tyb skulls are in bones of neural crest (NC) origin, and that mineralisation of the Dp1Tyb skull base synchondroses is aberrant. Furthermore, we show that increased dosage of Dyrk1a results in decreased NC cell proliferation and a decrease in size and cellularity of the NC-derived frontal bone primordia. Thus, DS craniofacial dysmorphology is caused by an increased dosage of Dyrk1a and at least three other genes

Comprehensive phenotypic analysis of the Dp1Tyb mouse strain reveals a broad range of Down syndrome-related phenotypes

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish whether this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes, including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state, and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder

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

Craniofacial dysmorphology in Down syndrome is caused by increased dosage of Dyrk1a and at least three other genes

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), occurs in 1 in 800 live births and is the most common human aneuploidy. DS results in multiple phenotypes, including craniofacial dysmorphology, which is characterised by midfacial hypoplasia, brachycephaly and micrognathia. The genetic and developmental causes of this are poorly understood. Using morphometric analysis of the Dp1Tyb mouse model of DS and an associated mouse genetic mapping panel, we demonstrate that four Hsa21-orthologous regions of mouse chromosome 16 contain dosage-sensitive genes that cause the DS craniofacial phenotype, and identify one of these causative genes as Dyrk1a. We show that the earliest and most severe defects in Dp1Tyb skulls are in bones of neural crest (NC) origin, and that mineralisation of the Dp1Tyb skull base synchondroses is aberrant. Furthermore, we show that increased dosage of Dyrk1a results in decreased NC cell proliferation and a decrease in size and cellularity of the NC-derived frontal bone primordia. Thus, DS craniofacial dysmorphology is caused by an increased dosage of Dyrk1a and at least three other genes.</p

Dosage-sensitive region causing locomotor dysfunction.

<p>Diagram on left shows Hsa21 indicating short and long arms separated by the centromere (oval), banding structure and length in Mb. The orthologous region of Mmu16 is indicated in grey and the regions of Mmu16 duplicated in Ts1Rhr, Dp4Tyb, Dp5Tyb and Dp6Tyb mouse models are indicated in black. On the right these duplicated regions are expanded and all known protein coding genes in these intervals and two microRNA genes (<i>Mir802</i> and <i>Gm23062</i>) are listed (mouse genome assembly GRCm38.p4). The locomotor defect assayed by Rotarod maps to a minimal interval resulting from the overlap of Ts1Rhr, Dp4Tyb and Dp5Tyb with genes in the interval indicated in blue. The <i>Dyrk1a</i> gene (bold) is required in 3 copies for the locomotor defect. Genes outside this region are listed in black.</p

Decreased numbers of motor neurons in Tc1 and Dp(16)1Yey mice.

<p>(<b>a</b>-<b>e</b>) Analysis of Tc1 mice and WT littermate controls at the indicated ages (n = 4 WT, 5 Tc1 at 4 months; 4 WT, 5 Tc1 at 19 months). Maximum force production by (<b>a</b>) TA, (<b>b</b>) EDL, and (<b>c</b>) Soleus muscles in response to tetanic stimulation. (<b>d</b>) Typical motor unit recording traces in WT and Tc1 mice, showing stepwise increments in single twitch force production in response to increased stimulating intensity. The number of increments recorded is taken as the number of motor units. (<b>e</b>) Number of motor units in EDL muscles of indicated mice. (<b>f</b>) Nissl-stained lumbar spinal cord sections of WT and Tc1 mice at 19 months of age, with the sciatic motor pool indicated by a dashed line. Inset shows motor neurons in the sciatic pool. Scale bars, 100 μm, inset 50 μm. (<b>g</b>) Number of spinal cord motor neurons in WT and Tc1 mice at 6 months (n = 8 WT, 5 Tc1) and 19 months of age (n = 4 WT, 5 Tc1). (<b>h</b>) Sections of the EDL, TA and soleus muscles of WT and Tc1 mice at 19 months of age, stained for succinate dehydrogenase (SDH) at low (left) and high (right) magnification. Muscle fibers with dark blue staining have higher SDH activity and thus rely on oxidative phosphorylation and are more likely to be slow fibers. Clustering of darker fibers in Tc1 mice indicates denervation and subsequent reinnervation by slow motor neurons and a shift towards slower muscle fibers. Scale bars: low magnification, 500 μm; high magnification, 50 μm. (<b>i</b>) Number of motor neurons in WT and Tc1 mice at 22d of age (n = 4 WT, 6 Tc1). (<b>j, k</b>) Number of motor neurons in mice of the indicated genotypes and matched controls at 6 months of age (n = 6 WT, 6 Dp(16)1Yey, 8 Dp(16)1Yey/Dp(10)1Yey/Dp(17)1Yey triple trisomic; 7 WT, 7 Dp(10)1Yey; 6 WT, 6 Dp(17)1Yey). (<b>l</b>) Number of motor neurons in Dp(16)1Yey mice and controls at P6 (n = 5 WT, 5 Dp(16)1Yey). (<b>m</b>) Number of motor neurons in mice of the indicated genotypes and matched controls at 6 months of age (n = 6 WT, 7 Dp9Tyb; 7 WT, 7 Dp2Tyb; 8 WT, 10 Dp3Tyb). All graphs show mean±SEM. Data analyzed with unpaired t-test, *p < 0.05, **p < 0.001, ***p < 0.0001.</p

No broad defect in sensory behavior in Tc1 or Dp1Tyb mice.

<p>Analysis of Tc1 (<b>a</b>-<b>e</b>) and Dp1Tyb (<b>f-k</b>) mice at 14 weeks (<b>a-d</b>, <b>f-j</b>), or 11 weeks (<b>e,k</b>). (<b>a,f</b>) Withdrawal latency of right and left hind paws in response to cold plate. (<b>b,g</b>) Withdrawal latency of right and left hind paws in response to radiant heat (Hargreaves test). (<b>c,h</b>) Force required for 50% withdrawal of right and left hind paws in response to punctate mechanical stimulation (Von Frey test). (<b>d,i</b>) Number of nocifensive behaviors as a function of time following formalin injection into hind paw. (<b>e</b>,<b>k</b>) Quantification of sensory neuron subpopulations in dorsal root ganglia taken from L3-L5 region of (<b>e</b>) Tc1 mice and (<b>k</b>) Dp1Tyb mice showing the proportion of DRG cell profiles positive for the indicated markers. (<b>j</b>) Score representing ability of WT or Dp1Tyb mice to walk up a tapered inclined beam; a higher score indicates better performance. All graphs show mean±SEM (<b>a-d</b>, n = 10; <b>e</b>, n = 5; <b>f-j</b>, n = 7; <b>k</b>, n = 5). Data analyzed with unpaired t-test, *p < 0.05.</p

Normal cerebellar anatomy in Dp(16)1Yey mice.

<p>(<b>a</b>-<b>e</b>) Analysis of cerebellar anatomy in Dp(16)1Yey mice at P6. (<b>a</b>) Cross section through cerebellum stained with H&E with expanded view showing small purple granule cells in the external granule layer (EGL) (arrow), Roman numerals indicate lobules, (<b>b</b>) area of cerebellum as % of whole brain, (<b>c</b>) width of the EGL, (<b>d</b>) linear density of Purkinje cells, (<b>e</b>) density of granule cells in the EGL. (<b>f</b>-<b>l</b>) Analysis of cerebellar anatomy in Dp(16)1Yey mice at 6 months of age. (<b>f</b>) Cross section through cerebellum stained with H&E with expanded view showing small purple granule cells in the granule cell layer (GCL) (arrow), Roman numerals indicate lobules, (<b>g</b>) linear density of Purkinje cells, (<b>h</b>) density of granule cells in the GCL, (<b>i</b>) width of the GCL, (<b>j</b>) width of the molecular layer (ML), (<b>k</b>) cross section of lobule IX showing the ML subdivided into base, tip and inner and outer regions, (<b>l</b>) density of interneurons in the whole ML of lobule IX or in the base, tip or inner regions subdivided as in <b>k</b>. In <b>b</b>-<b>d</b> and <b>g</b>-<b>j</b> values are shown for individual lobules as indicated and also averaged over all lobules analyzed (n = 9 WT, 13Ts(16)1Yey P6 whole brain area; n = 7 WT, 12 Dp(16)1Yey at P6 for all other measurements; n = 9 WT and Dp(16)1Yey at 6 months). All graphs show mean±SEM.</p

Increased <i>Dyrk1a</i> expression in Dp(16)1Yey and Dp3Tyb mice.

<p>(<b>a-d</b>) Mean±SEM mRNA levels of <i>Dyrk1a</i>, <i>Gad1</i> and <i>Gad2</i> in the cerebellum of Dp(16)1Yey mice at 10 weeks (<b>a</b>) and 6 days (<b>b</b>) of age and in 10 week old Dp3Tyb (<b>c</b>) and Dp5Tyb (<b>d</b>) mice. Expression of the test gene was normalized to <i>Gapdh</i> and then to expression in WT control mice (n = 5 of each genotype). Data analyzed with unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001.</p