Summary
Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (
AD
) thrombocytopenia with normal platelet ...size. Linkage analysis and whole exome sequencing identified the R1026W substitution in
ITGA
2B
as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with
AD
thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with
AD
thrombocytopenia with normal platelet size resulting from mutation in
ITGA
2B
.
ITGA
2B
mutations should therefore be included in the differential diagnosis of this latter disorder.
SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII ...vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A-D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss of SEC24C. Conditional KO of Sec24c in neural progenitors during embryogenesis caused perinatal mortality and microcephaly, with activation of the unfolded protein response and apoptotic cell death of postmitotic neurons in the murine cerebral cortex. The cell-autonomous function of SEC24C in postmitotic neurons was further highlighted by the loss of cell viability caused by disrupting Sec24c expression in forebrain neurons of mice postnatally and in differentiated neurons derived from human induced pluripotent stem cells. The neuronal cell death associated with Sec24c deficiency was rescued in knockin mice expressing Sec24d in place of Sec24c. These data suggest that SEC24C is a major cargo adapter for COPII-dependent transport in postmitotic neurons in developing and adult brains and that its functions overlap at least partially with those of SEC24D in mammals.
Summary
Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet ...size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with AD thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with AD thrombocytopenia with normal platelet size resulting from mutation in ITGA2B. ITGA2B mutations should therefore be included in the differential diagnosis of this latter disorder.
Congenital Dyserythropoietic Anemia type II (CDAII) is a disease of ineffective erythropoiesis characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroid precursors. ...CDAII is an autosomal recessive disease resulting from mutations in SEC23B. SEC23 is a core component of COPII vesicles, which transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi. Despite identification of the underlying genetic defect, the molecular mechanism by which SEC23B deficiency produces the unique CDAII phenotype remains unknown. We previously reported that mice homozygous for a Sec23b null allele die perinatally, exhibiting massive pancreatic degeneration, precluding evaluation of the adult erythroid compartment. To examine the impact of SEC23B deficiency on adult murine hematopoiesis, we generated mice with erythroid specific and pan-hematopoietic deficiency for SEC23B by crossing a second, conditional Sec23b allele (Sec23bfl), in which exons 5 and 6 are flanked by loxP sites, to an EpoR-Cre and Vav1-Cre, respectively. These mice did not exhibit anemia or any other CDAII characteristic. Similarly, mice transplanted with SEC23B-deficient fetal liver cells harvested from E17.5 embryos also failed to recapitulate any features of the CDAII phenotype. We next generated mice deficient in SEC23B exclusively in the pancreas by crossing the Sec23bfl allele to either p48-Cre or Pdx1-Cre. These mice exhibit a phenotype indistinguishable from mice with germline deletion of Sec23b, indicating that loss of pancreatic SEC23B is sufficient to explain the perinatal-lethality of global SEC23B deficiency in mice. The mammalian genome contains two paralogs for SEC23, SEC23A and SEC23B. These paralogs are highly identical at the amino acid level (~85%). We examined the relative expression of SEC23B/SEC23A in WT tissues from both humans and mice. This ratio is higher in human BM compared to pancreas, while it was higher in mouse pancreas compared to BM. In order to determine if SEC23A can rescue the phenotype of SEC23B deficient mice when expressed under the regulatory control of Sec23b, we genetically engineered the Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23a-b) in mouse embryonic stem cells via recombinase mediated cassette exchange. A heterozygous Sec23a-b intercross yielded the expected number of mice homozygous for the Sec23a-b allele. These mice exhibited normal survival, development, and fertility. Pancreas tissues dissected from Sec23a-b/a-b mice had normal weights, were histologically indistinguishable from WT controls, and did not exhibit dilated ER by transmission electron microscopy. Western blot analysis confirmed the absence of SEC23B in pancreata of Sec23a-b/a-b mice, with high levels of SEC23A expression. These data demonstrate that the SEC23A and SEC23B proteins overlap significantly (or completely) at the level of protein function, and suggest that the distinct phenotypes of human and mouse SEC23 deficiency are the result of an evolutionary shift in the tissue-specific gene expression programs of SEC23A and SEC23B. These findings also suggest that therapies that increase the expression of either SEC23 paralog in erythroid cells might be effective in CDAII.
No relevant conflicts of interest to declare.
Combined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder resulting from mutations in Lman1. This gene encodes a cargo receptor in the early secretory ...pathway that is responsible for the efficient secretion of factor V (FV) and factor VIII (FVIII) to the plasma. F5F8D is characterized by levels of both FV and FVIII reduced to ∼5-30% of normal. In contrast, Lman1 knockout mouse models of F5F8D exhibit FV and FVIII activities that are ∼50% of normal, relative to wildtype mice. Though FV and FVIII are synthesized at markedly different levels and potentially in different tissues, loss of the LMAN1 cargo receptor leads to parallel reductions in both FV and FVIII activity. FV is synthesized in hepatocytes (as well as megakaryocytes in the mouse). However, the primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. We took advantage of the dependence of efficient FV and FVIII secretion on LMAN1 to examine the cellular source of each protein. FV and FVIII secretion profiles of conditional Lman1 knockout mice were characterized, relative to that of wildtype mice and ubiquitous Lman1 null mice (Lman1-/-). In order to generate mice with Lman1 expression specifically deleted in the endothelium or the hepatocytes, either a Tie2-Cre or Albumin-Cre transgene was crossed into Lman1 conditional mice (Lman1fl). FV and FVIII activity levels were measured by functional coagulation activity assays. Though Lman1fl/fl/Tie2-Cre+ mice (endothelial-specific knockout) exhibit normal plasma FV activity (99.9%) relative to wildtype mice (set to 100%), FVIII activity is reduced to 53.5% (p < 2.5 x 10-6). In contrast, Lman1fl/fl/Alb-Cre+ (hepatocyte-specific knockout) mice demonstrate normal FVIII activity (89.0%) and reduced FV activity (37.0%) (p < 1.4 x 10-10). To confirm endothelial cells as the biosynthetic source of FVIII, we took advantage of the previously reported RiboTag mouse (Sanz et al., 2009. PNAS 106(33):13939-44) to isolate endothelial cell RNA for qPCR analysis from various murine tissues. RiboTag mice carry a hemaglutinin-tagged ribosomal protein that can be used for cell-type specific immunoprecipitation of polyribosomes and subsequent RNA analysis when crossed with a Cre-recombinase expressing animal. qPCR analyses of endothelial cell RNA isolated from total liver lysates of five RiboTag/Tie2-Cre+ mice demonstrated 10-20 fold enrichment for gene transcripts that are known to be endothelial-specific, such as Cdhs (12.1 fold enrichment, p < 8.0 x 10-3), Vcam1 (13.4 fold enrichment, p <1.1 x 10-5), and Vwf (15.3 fold enrichment, p < 7.0 x 10-4), as well as for FVIII transcripts (11.4 fold enrichment, p < 4.0 x 10-5). In contrast, this analysis demonstrated a statistically significant depletion (5-10 fold) of transcripts from many known hepatocyte-specific genes, including multiple coagulation factor genes. Similar examination of kidney endothelial cell RNA also demonstrated enrichment for FVIII transcripts, thereby demonstrating that endothelial cells from multiple tissues and vascular beds contribute to the plasma FVIII pool in the mouse. These results explain the successful reversal of hemophilia A by both liver and kidney transplants. Taken together, these results definitively demonstrate that endothelial cells are the primary source of FVIII biosynthesis in the mouse, and that hepatocytes make no significant contribution to the plasma FVIII pool.
No relevant conflicts of interest to declare.
Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disease characterized by anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from loss ...of function mutations in SEC23B encoding a core component of coat complex protein II (COPII) vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Despite the identification of the genetic cause of CDAII, the pathophysiology of the disease remains unknown.
Morpholino-induced SEC23B deficiency in zebrafish (ZF) has been previously reported to result in an erythroid phenotype mimicking CDAII (Shwartz et al, Nature genetics 2009), suggesting conservation of the underlying CDAII mechanism from fish to humans. Thus, we were puzzled to observe the absence of anemia or other CDAII characteristics in mice with erythroid specific (EpoR -Cre) and pan-hematopoietic (Vav1 -Cre) SEC23B deficiency (Khoriaty et al, MBC and Khoriaty et al, Sci Rep).
To re-examine the ZF phenotype, we injected the morpholino targeting Sec23b into one-cell stage ZF embryos demonstrating no increase in circulating bi-nucleated erythroid cells, in contrast to the previous report. Given the variable knock-down that can result from morpholinos, we next generated ZF heterozygous for a 53 bp deletion (Sec23b+/-) using CRISPR/Cas9 genome editing. Intercrosses between Sec23b+/- ZF demonstrated lethality of Sec23b-/- ZF between days 17-21. However, the percentage of circulating bi-nucleated erythrocytes observed at day 16 was indistinguishable between Sec23b-/- ZF and wildtype (WT) clutch mate controls.
Mammals and fish express two paralogs for SEC23, SEC23A and SEC23B, encoding highly related (~85%) proteins. To investigate the different functions of SEC23A and SEC23B, we defined the SEC23A and SEC23B interactomes using “BioID” (proximity dependent biotinylation) in HEK293 cells expressing BirA*-tagged SEC23A, SEC23B, or GFP control. Surprisingly, SEC23A and SEC23B exhibit indistinguishable interactomes. We also demonstrated that both mouse and human SEC23 paralogs can complement SEC23 deficiency in yeast. Similarly, rescue of the Sec23b-/- lethal phenotype in ZF by a Sec23a transgene demonstrated at least partial functional overlap of SEC23A/SEC23B function in vertebrates.
To extend these observations to mammals, we genetically engineered the murine Sec23a cDNA into the endogenous mouse genomic locus of Sec23b . We demonstrated that SEC23B-deficient mice (previously shown to die perinatally from pancreatic degeneration) are rescued by SEC23A, exhibiting normal survival and pancreas histology, with no abnormalities apparent on detailed hematologic and anatomic examination.
The expression of SEC23A and SEC23B mRNAs in human and mouse BMs were examined by qRT-PCR. SEC23B is the predominantly expressed paralog in human BM, with greater levels of SEC23A and reduced SEC23B in mouse BM. We therefore hypothesized that mice with erythroid deficiency of SEC23A alone or combined SEC23A/SEC23B deficiency might exhibit an erythroid defect. We first generated mice with erythroid-specific SEC23A deficiency, with the latter mice exhibiting no anemia or other CDAII characteristic. In contrast, mice with combined erythroid SEC23A and SEC23B deficiency die at ~E12.5, exhibiting reduced size and appear white in color compared to their WT litter mate controls, consistent with requirement of SEC23 in the erythroid compartment.
Taken together, these data suggest complete (or near complete) overlap in function between SEC23A and SEC23B, and suggest that therapies that increase the expression of either SEC23 paralog might prove effective in treating CDAII. This paradigm might also apply to other disorders due to mutations in paralogous genes. Finally, our findings also suggest that a switch in paralog expression could account for other disparate disease phenotypes observed between animal models and humans.
No relevant conflicts of interest to declare.
T cells play critical roles in both protective and pathogenic adaptive immune responses. The release of cytokines by T cells is critical for their development, differentiation, and effector ...functions. However, significant gaps remain in our understanding of the fundamental cellular and molecular pathways that are critical for T cell cytokine release. Coat Protein Complex II (COPII) enables protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. It consists of an outer layer that facilitates the budding of vesicles from the ER membrane, and an inner layer responsible for selecting cargo proteins that comprises the protein SEC23B. The critical ER-Golgi pathway in T cell cytokine release is not known.
Therefore, to explore the role of COPII secretory pathways, we generated novel Sec23bfl/-Cd4Cre mice that will lack Sec23b in T cells. We hypothesized that SEC23B-mediated COPII deficiency will lead to abnormalities in T cell development given the critical roles of T cell-derived cytokines in their ontogeny. Contrary to our hypothesis, SEC23B-deficient T cells developed normally, and were present in similar numbers in the thymus (p=0.20), lymph nodes (p=0.69), and spleen (p=0.10) as WT T cells. They also showed normal levels of surface phenotypic markers (TCR, CD25, CD44, CD62L, CD28). To rule out potential Cre-recombinase driven artifacts we next generated Sec23bfl/-Vav1Cre mice and found that T cell development was not affected. We explored the functions of mature fully developed T cells by stimulating them with aCD3/aCD28 for 3 days. Compared to WT T cells, SEC23B-deficient T cells demonstrated decreased release of cytokines such as IL-2, IL-7, and IFNg (all p<0.01), but not others, such as IL-17 and IL-10. This selective reduction in cytokine secretion was not due to defective T cell signaling events (such as phosphorylation of ZAP-70, LCK, AKT, ERK1/2) or production of cytokines at the mRNA level (including IL-2, IFNg, IL-10). However, the altered cytokine levels in the supernatant was associated with decreased proliferation by SEC23B-deficient T cells compared to WT when measured by CFSE (p=0.0003).
We therefore hypothesized that SEC23B-deficient T cells were able to produce cytokines, but unable to release them selectively. To test this, we used Brefeldin A (BFA), an inhibitor of anterograde transport from the ER to the Golgi apparatus. In the presence of BFA, intracellular levels of IL-2 and IL-17 were similar in WT and SEC23B-deficient T cells (p=0.86). Collectively, these data suggest SEC23B-deficient T cells exhibit secretory defects of select cytokines upon stimulation compared to WT T cells.
We next explored the in vivo relevance of SEC23B-dependent COPII vesicle formation in two clinically relevant models. We first tested the impact of disrupting Sec23b on in vivo protective functions of T cells in a model of viral immunity by utilizing the well-characterized LCMV Armstrong model of acute infection. Sec23b WTor Sec23bfl/-Cd4Cre mice were infected with 2x105 p.f.u. of LCMV Armstrong i.p., and T cells were analyzed on day 8 post-infection. In contrast to WT mice, Sec23bfl/-Cd4Cre mice demonstrated impaired ability to clear the virus by day 8 (9.1x107 p.f.u/gm vs N.D., p=0.0008), reduced expansion of viral antigen-specific cells (p=0.0001 for gp33, gp66, p=0.0006 for gp276), and reduced numbers of IFNg (p=0.0004) and TNFa (p=0.0003) producing T cells on day 8 post-infection.
We next determined the pathogenic capacity of SEC23B-deficient T cells by utilizing a well-established MHC-mismatched C57BL/6->BALB/c model of Graft-versus-Host Disease (GVHD). BALB/c and C57BL/6 recipients were lethally irradiated (8.5 Gy) and transplanted with 5x106 allogeneic C57BL/6 WT bone marrow cells, and either 2x106 C57BL/6 WT or SEC23B-deficient T cells. All syngeneic recipients survived. By contrast, 100% of the recipients of allogeneic WT T cells died by day 61 due to GVHD. However, only 6% of allogeneic recipients of SEC23B-deficient T cells died (n=18/group, p<0.0001) and all recipients showed lower GVHD clinical scores compared to recipients of WT T cells (p<0.01 to 0.001 over 6 weeks).
Collectively, our data demonstrate a heretofore unrecognized but critical role for SEC23B-dependent COPII in T cell protective and pathogenic effector functions and thus provide fundamental insights into T cell-mediated immunity.
Ginsburg:Portola pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire plc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell-specific
deletion. Inactivation of
...exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar
deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice.
SEC23B mutations in humans result in the autosomal recessive disease Congenital Dyserythropoietic Anemia type-II (CDAII). CDAII is characterized by moderate anemia in increased bone marrow (BM) ...bi/multi-nucleated erythroblasts. Despite the identification of the genetic defect underlying CDAII, the pathophysiology of this disease remains unknown. SEC23A and SEC23B are paralogous components of the coat protein complex II (COPII)-coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum to the Golgi apparatus. We generated SEC23B-deficient mice and demonstrated that these animals die perinatally exhibiting massive pancreatic degeneration (Tao et. al PNAS). To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, we harvested fetal liver cells (FLC), which contain hematopoietic stem cells, from SEC23B-deficient or wild-type (WT) control E17.5 embryos and transplanted them into lethally irradiated C57BL/6J mice. Recipients of SEC23B-deficient FLC did not exhibit anemia or any other CDAII characteristic (Khoriaty et. al, Mol Cell Biol), and SEC23B deficient FLC competed effectively with WT FLC at reconstituting hematopoiesis when transplanted into lethally irradiated recipient mice (Khoriaty et. al, Mol Cell Biol). A Sec23bfl conditional-allele was also generated. Mice with hematopoietic-specific SEC23B-deficiency, generated by crossing Vav1-Cre into Sec23bfl mice, also exhibited normal hematopoiesis. In contrast, mice with pancreas-specific Sec23b deficiency generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre resulted in a phenotype indistinguishable from complete SEC23B-deficiency, demonstrating that loss of pancreatic Sec23b expression is sufficient to explain the perinatal lethality of SEC23B-deficient mice. To investigate different phenotypes of SEC23B deficiency in humans and mice, the SEC23B/SEC23A expression ratio was examined in murine and human tissues. This ratio is higher in mouse pancreas (12.7) compared to BM (2.6), whereas it is higher in human BM (7.8) relative to pancreas (5.5). Taken together with the high degree of amino-acid identity between SEC23A and SEC23B (~85%), these data suggest that the tissue-specific functions of SEC23A and SEC23B have shifted during evolution between humans and mice. To determine if Sec23a can rescue the lethality of SEC23B-deficient mice, we have engineered Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23A-B) via recombinase-mediated cassette exchange. A heterozygous Sec23A-B intercross is in progress. Rescue of the SEC23B deficient phenotype by SEC23A protein expressed under control of Sec23b regulatory sequences would suggest that increasing the expression of either paralog in erythroid cells might be effective in the treatment of CDAII.
No relevant conflicts of interest to declare.
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