conditional hematopoietic dnmt3a knockout mice  (Jackson Laboratory)

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) DNMT3A mutation rates are shown for the gnomAD and SPARK control databases individually, and for the SPARK and gnomAD databases combined. Mutation rates are also shown for PAH, IPAH and NON-IPAH patients. Mutation rate is shown on the y-axis of the graph (in %) and is displayed for the 3 control cohorts and 3 PAH cohorts . B) Fisher’s exact test was used to compare the mutation rate between PAH, SPARK and gnomAD databases. Fisher’s exact test p-values as well as odds ratios and the associated confidence intervals (95% confidence) are shown for several PAH vs control comparisons. Mutation rates were significantly increased in the PAH Biobank (1%) compared to both SPARK (0.33%; p=<0.0001) and gnomAD (=> 0.43%; p = 0.0002) controls. Mutation rates were also significantly increased in both total PAH patients and non-IPAH patients (1.64%) compared to both control databases combined (0.42%, p=0.0003 and 0.0002, respectively). There was no significant difference between the SPARK and gnomAD control databases (p=0.1133). C) The age groups of the cohorts were more comparable between gnomAD and PAH databases. % of the cohort (y-axis) at each age range (x-axis) is shown for the PAH Biobank as well as both control databases.

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Mutagenesis

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet:

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques:

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) Data was obtained using deep, targeted panel sequencing. The percentage of individuals with CHIP is shown for PAH patients (n=710) and controls (n=3645). Approximately 15% of PAH patients possessed a CHIP mutation while only 7% of controls were found to have CHIP. B) The number of patients with CHIP mutations in each gene is displayed. Number of patients is shown on the y-axis of the graph while genes are shown on the x-axis. DNMT3A was the most mutated gene (red; 49 patients), followed by TET2 (green, 18 patients). All other mutated genes are shown in purple. The pie chart shows the mutation distribution using the same colours and displays the number of patients with no CHIP mutations in blue. C) The prevalence of all CHIP mutations, DNMT3A CHIP and non- DNMT3A CHIP is shown. 104/710 PAH patients were found to have CHIP mutations (49 being in DNMT3A , and 55 in other genes). The odds ratios, with 95% confidence intervals, are also shown for PAH patients with all CHIP mutations, DNMT3A CHIP and non- DNMT3A CHIP compared to controls. The odds ratios for all CHIP mutations and non- DNMT3A CHIP mutations were statistically significant (p=0.0071, p=0.002, respectively), though DNMT3A CHIP alone did not reach statistical significance (p=0.40).

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Sequencing, Mutagenesis

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) The three DNMT3A transcript variants 2 (NM_153759.3), 3 (NM_022552), and 4 (NM_175630.1), which code for the three protein isoforms DNMT3A2, DNMT3A3, and DNMT3A4, respectively, are shown, in comparison to the original DNMT3A transcript variant 1. B-D) Gene expression of the DNMT3A isoforms 2,3 and 4 was measured in PBMCs. Decreased expression of variant 4 in 88% of SSc-PAH and 93.3% of IPAH patients (IPAH 0.77; SSc-PAH 0.8; p<0.0001), as well as decreased expression of variant 3 in 80% of SSc-PAH and 73.3% of IPAH patients compared to healthy individuals (IPAH 0.92; SSc-PAH 0.93; p<0.05) was found. The gene expression of variant 2 was unaffected. E-G) Receiver operating characteristic (ROC) analysis on a cohort consisting of 41 healthy controls and 80 PAH patients (IPAH/SSc-PAH) was performed for DNMT3A isoforms 2, 3 and 4. Sensitivity % is shown on the y-axis while the x-axis represents 100% subtract the Specificity %. The results indicate that DNMT3A variant 4 (AUC: 0.82; p<0.0001) and variant 3 (AUC: 0.67; p<0.002) could serve as potential predictors of PAH.

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Comparison, Variant Assay, Expressing

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: A) Hematopoietic Dnmt3a -knockout mice spontaneously develop PAH by 9 months of age (n=4-5/group). Right ventricular systolic pressure (RVSP; in mmHg) and mean pulmonary arterial pressure (mPAP; in mmHg) are significantly increased in Dnmt3a -knockout mice (green) compared to controls (white; p=0.0004, p=0.0004, respectively). Pulmonary artery acceleration time (PAAT; in ms), tricuspid annular plane systolic excursion (TAPSE; in mm) and cardiac output (CO; in ul/min) are significantly reduced in Dnmt3a -knockout mice compared to controls (p=0.0003, p=0.012; p=0.006, respectively). Right, ventricular end-diastolic pressure (RVEDP; in mmHg) shows a trend towards being increased in Dnmt3a -knockout mice, though not statistically significant. Right-heart catheterization (RHC) traces of RVSP are shown on the left for both control (top) and knockout (bottom) mice. RVSP, mPAP, and RVEDP were obtained via RHC, while PAAT, TAPSE and CO were obtained via echocardiography. B) Hematopoietic Dnmt3a -knockout mice develop PAH, accelerated by a second hit hypoxia (n=6-9/group). 3-month-old control (white boxes) and Dnmt3a -knockout (orange boxes) mice were either maintained in normoxic (solid colour boxes) conditions for 6 weeks or were exposed to 3 weeks of hypoxia followed by 3 weeks of normoxia (boxes with bricks). Representative RVSP traces obtained via RHC are shown for each group. RVSP, mPAP and RVEDP (trending) are elevated in Dnmt3a -knockout mice that underwent a second hit (hypoxia) compared to hypoxic controls (p=0.0004, p=0.0007, p=0.0724, respectively), and RVSP, mPAP and RVEDP are also significantly increased in Dnmt3a -knockout mice that were kept in normoxia compared to the normoxic controls (p=0.0015, p=0.0015, p=0.0234, respectively). PAAT and TAPSE are significantly reduced in Dnmt3a -knockout mice that underwent a second hit (hypoxia) compared to controls (p=0.0020, p=0.0222, respectively) and are also significantly increased in Dnmt3a -knockout mice that were kept in normoxia, compared to the normoxic controls (PAAT: p=0.0030; TAPSE: p=0.0001). CO was significantly reduced in hypoxic Dnmt3a -knockout mice compared to controls (p=0.0101), while normoxic Dnmt3a -knockout mice showed a trend towards being reduced, though not statistically significant (p=0.2061). * = p< 0.05. Treatment of hypoxic Dnmt3a -knockout mice with Canakinumab (yellow bars with bricks) improved hemodynamic measurements and cardiac function (n=3; RVSP: p=0.0362, mPAP: p=0.0449, PAAT: p=0.0258, TAPSE: p=0.0533, CO: p=0.0299). RVEDP was not significantly reduced (p=0.4629).

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Knock-Out

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: Histological assessment via hematoxylin and eosin (H&E) staining was performed on lung tissue slides and small pulmonary arteries were identified. Pulmonary medial wall thickness was compared between control and knockout mice. A) Pulmonary vascular remodelling occurs in the lungs of hematopoietic Dnmt3a -knockout mice by 9 months of age. There is a significant increase in the pulmonary medial wall thickness, defined as the % of the wall consisting of tunica media, in hematopoietic Dnmt3a -knockout mice (green) compared to controls (white; p=0.0005; n=5/group). A t-test was used to assess the difference between control and knockout mice. (B) There is an increase in pulmonary medial wall thickness in the lungs of hematopoietic Dnmt3a -knockout mice exposed to a second-hit hypoxia compared to controls (p<0.0001; n=3/group). 4.5-month-old Dnmt3a -knockout mice also spontaneously developed pulmonary vascular remodeling (p<0.0001; n=3/group). Bars with bricks represent hypoxic exposure while solid bars represent normoxia only. White represents control mice and knockout mice values are shown in orange. (C) Collagen deposition assessment of RV tissue using Picrosirius red staining (n=3/ group) demonstrated increased collagen deposition in the RV tissue of both hypoxic and normoxic Dnmt3a -knockout mice compared to their respective controls.

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Staining, Knock-Out

Journal: medRxiv

Article Title: Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH

doi: 10.1101/2023.12.30.23300391

Figure Lengend Snippet: Immunofluorescence and confocal microscopy were used to measure leukocyte infiltration in the lungs of Dnmt3a -knockout mice with PAH compared to controls. CD45, a marker of leukocytes, is shown in green. DAPI, a nuclei stain, is shown in blue. (A) There is a trend towards an increase in the number of CD45+ cells in the lungs of nine-month-old knockout mice (green), that have developed PAH, compared to controls (white; n=2/group). The graph represents the number of CD45+ cells per 0.02 mm . (B) There is a significant increase in the number of CD45+ cells in the lungs of the knockout mice, that have developed PAH, compared to controls (n=5/group, p=0.0473). Hypoxia appears to exacerbate leukocyte infiltration of the lungs, though there was no significant difference between knockout and control mice in hypoxia. Bars with bricks represent 3 weeks of hypoxic exposure with 3 weeks of normoxic exposure while solid bars represent 6 weeks of normoxia. White bars represent control mice and orange bars represent Dnmt3a -knockout mice. (C) Flow cytometry was used to investigate the subpopulations of leukocytes seen on confocal microscopy. Following a live/dead stain, CD45+ cells (leukocytes) were gated for and were found to be increased on 4.5-month-old Dnmt3a -knockout mice compared to controls (n=5-8/group; p=0.05). Specifically, macrophages are increased in Dnmt3a -knockout mice (n=5-8/group; p=<0.01). An LY6G antibody was used for neutrophil detection, an F4/80 antibody was used for macrophage detection, a CD3 antibody was used for T-cell detection, and a CD19 antibody was used for B-cell detection. On average, approximately 90% of CD45+ cells in the control mice were macrophages, while this was increased to approximately 95% in the Dnmt3a -knockout mice. D) IL-13 was found to be increased in the plasma of Dnmt3a -knockout mice compared to controls (p=0.05). There was also a trend towards an increase in plasma G-CSF in the Dnmt3a -knockout mice compared to controls (p=0.16). Plasma cytokine concentration is shown on the y-axis in picogram per milliliter (pg/ml).

Article Snippet: We produced male and female conditional hematopoietic Dnmt3a knockout mice by crossing parental floxed and Vav-iCre mice as described by the Jackson Laboratory ( https://www.jax.org/strain/008610 ) and by Joseph et al. 3334 This knockout simulates the ‘loss of function’ effects of DNMT3A somatic mutations seen in patients with PAH in our U.S. PAH Biobank data.

Techniques: Immunofluorescence, Confocal Microscopy, Knock-Out, Marker, Staining, Flow Cytometry, Concentration Assay