Congenic Mice Confirm That Collagen X Is Required for Proper Hematopoietic Development

The link between endochondral skeletal development and hematopoiesis in the marrow was established in the collagen X transgenic (Tg) and null (KO) mice. Disrupted function of collagen X, a major hypertrophic cartilage matrix protein, resulted in skeletal and hematopoietic defects in endochondrally derived tissues. Manifestation of the disease phenotype was variable, ranging from perinatal lethality in a subset of mice, to altered lymphopoiesis and impaired immunity in the surviving mice. To exclude contribution of strain specific modifiers to this variable manifestation of the skeleto-hematopoietic phenotype, C57Bl/6 and DBA/2J collagen X congenic lines were established. Comparable disease manifestations confirmed that the skeleto-hematopoietic alterations are an inherent outcome of disrupted collagen X function. Further, colony forming cell assays, complete blood count analysis, serum antibody ELISA, and organ outgrowth studies established altered lymphopoiesis in all collagen X Tg and KO mice and implicated opportunistic infection as a contributor to the severe disease phenotype. These data support a model where endochondral ossification-specific collagen X contributes to the establishment of a hematopoietic niche at the chondro-osseous junction

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Number of LSK cells in collagen X moue bone marrow.

<p>Quantification of lineage^-, Sca-1⁺, cKit⁺ (LSK) cells in the bone marrow of week-3 wild type (WT), collagen X transgenic (Tg), collagen X null (KO) and the perinatal lethal collagen X Tg subset (Tg-severe) via flow cytometry. 10 mice per group. (**p<0.001)</p

Altered spleen architecture and B lymphocyte profile in the collagen X Tg and KO mice.

<p>Giemsa and immunohistochemical staining of spleens from week-3 C57Bl/6 wild type (A, 2X; B, Hi mag) and congenic collagen X perinatal lethal mice (C, 2X; D, Hi mag) revealed diminished organs with altered tissue architecture in the perinatal lethal mice. Staining for red blood cells with TER119 antibody (upper halves of spleens on right of Giemsa stained sections) showed decreased zones of red pulp (RP) in perinatal lethal mouse spleen. Staining for B lymphocytes with B220 antibody (lower halves of spleens on right of Giemsa stained sections) revealed diffuse B220⁺ staining and a reduction of lymphatic node (N) size in perinatal lethal mouse spleen. Bar = 25 mm. (E) Temporal analysis by flow cytometry for B220⁺ B lymphocytes from of outbred and congenic (C57Bl/6, B6 and DBA/2J, DBA) wild type (WT) and collagen X transgenic (Tg), null (KO), and perinatal lethal (Tg severe, KO severe) mouse spleens revealed decreased B lymphocytes throughout life, with the exception of KO at weeks two and three, in both the outbred and congenic strains. (F) Number of B220⁺ B lymphocytes in congenic WT, collagen X Tg and KO mice. Note depletion of B lymphocytes in Tg severe and KO severe. Numbers above the standard error of the mean are the number of mice per group. (*p<0.05, **p<0.01)</p

Increased microbial contamination during In vitro organ cultures from collagen X perinatal lethal mice.

<p>Isolated and cultured lung, liver and spleen from week-3 control (WT), collagen X transgenic and null (Col X Tg + KO, pooled data), and Tg and KO perinatal lethal (Col X severe; pooled data) mice reveal increased microbial contamination in perinatal lethal samples. Percent contamination was determined as number of wells contaminated with either bacteria or fungus after 24 hr culture, over the total number of wells per mice tested (in parenthesis).</p

Percent perinatal lethality in collagen X outbred and congenic mice.

<p>Conservation of perinatal lethality for the outbred and congenic (C57Bl/6 and DBA/2J) collagen X transgenic mice (1.6–293Δ and 4.7–21Δ) and collagen X null (KO) mice excluded contribution of a strain-specific modifier to the variable disease phenotype. Percent perinatal lethality was calculated as the number of wasting mice around week-3 (n) divided by the total number of weaned offspring given by mutant producing parents.</p

Collagen X Tg and KO mice have an altered chondro-osseous junction.

<p>Hematoxylin and eosin staining of longitudinal sections of tibia from (A) week-3 C57Bl/6 wild type (WT), (B) C57Bl/6 congenic collagen X transgenic (Tg), (C) null (KO), and perinatal lethal (D) Tg, and (E) KO. The chondro-osseous junction (COJ) is shown, including hypertrophic cartilage (HC), trabecular bone (TB), and marrow (M). Note diminished trabecular bony spicules in all collagen X Tg and KO mice, with the greatest reductions in (D) and (E), with a concomitant increase in erythrocytes in the marrow. Bar = 150 µm.</p

Collagen X perinatal lethal mice have increased levels of serum antibodies.

<p>Sera from week-3 wild type (WT), collagen X transgenic (Tg), null (KO) and perinatal lethal (Tg severe and KO severe; pooled) mice were assayed for IgM and IgG antibodies with ELISA. (A-B) Total IgM and IgG serum antibody measured. (C-D) Normalized levels of IgM and IgG antibodies calculated as (ng/ml of serum antibody)/(total number of live B220 splenocytes). Numbers above the standard error of the mean are number of mice per group.</p

Colony forming cell assays confirm altered hematopoietic lineage commitment in the collagen X Tg and KO mice.

<p>Colony forming assays using marrow aspirates from week-3 wild type (WT), collagen X transgenic (Tg), null (KO) and perinatal lethal (Tg severe and KO severe) mice. Cells cultured in: (A-B) complete Methocult media to enumerate granulocyte-macrophage (GM) and granulocyte-erythrocyte-monocyte-macrophage (GEMM) colonies; (C) erythropoietin supplemented Methocult media to enumerate pre-erythrocyte blast colonies (BFU-E); (D) interluekin-7 supplemented Methocult media to enumerate pre-B lymphocyte colonies (pre-B). Numbers above the standard error of the mean are number of samples. (*p<0.05, **p<0.001).</p

Diminished number of immature thymocytes and altered thymic architecture in the collagen X perinatal lethal mice.

<p>(A, B) Giemsa staining of thymuses from week-3 wild type (WT) and collagen X transgenic perinatal lethal (PL) mice reveals a reduction in size and depletion of the cortical thymocytes and region in the perinatal lethal mouse thymus, (2X) Bar = 775 µm and 20X respectively. (C) Flow cytometry analysis of week-3 C57Bl/6 wild type, congenic collagen X transgenic (Tg), null (KO) and perinatal lethal (Tg severe and KO severe) mouse thymocytes quantifies the percent double-positive, CD4⁺/CD8⁺, immature T lymphocytes (boxed). (D) Graphic representation of compiled flow cytometry data reveals decreased levels of CD4⁺/CD8⁺ immature T lymphocytes in perinatal lethal collagen X Tg and KO mice across all strains (outbred; OB, C57Bl/6; B6, and DBA/2J; DBA). n≥6 mice per group.</p