Rami Khoriaty

Murine coagulation factor VIII is synthesized in endothelial cells

The primary cellular source of factor VIII (FVIII) biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. LMAN1 is a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and FVIII to the plasma. Lman1 mutations result in combined deficiency of FV and FVIII, with levels of both factors reduced to ~10% to 15% of normal in human patients. We generated Lman1 conditional knockout mice to characterize the FVIII secretion profiles of endothelial cells and hepatocytes. We demonstrate that endothelial cells are the primary biosynthetic source of murine FVIII and that hepatocytes make no significant contribution to the plasma FVIII pool. Utilizing RiboTag mice and polyribosome immunoprecipitation, we performed endothelial cell-specific messenger RNA isolation and quantitative polymerase chain reaction analyses to confirm that endothelial cells highly express F8 and to explore the heterogeneity of F8 expression in different vascular beds. We demonstrate that endothelial cells from multiple, but not all, tissues contribute to the plasma FVIII pool in the mouse.

The changing face of neurosyphilis

The incidence of syphilis has increased over the past decade, particularly among HIV-positive patients, and the presenting clinical features have changed since the beginning of the HIV epidemic. The clinical manifestations of neurosyphilis are protean, and include acute stroke. In patients with HIV, the diagnosis and treatment of neurosyphilis is challenging. We review the clinical presentation, pathophysiology, and treatment of neurosyphilis, with emphasis on neurosyphilis in the HIV population, and neurosyphilis as a cause of acute stroke.

The COPII pathway and hematologic disease

Multiple diseases, hematologic and nonhematologic, result from defects in the early secretory pathway. Congenital dyserythropoietic anemia type II (CDAII) and combined deficiency of coagulation factors V and VIII (F5F8D) are the 2 known hematologic diseases that result from defects in the endoplasmic reticulum (ER)-to-Golgi transport system. CDAII is caused by mutations in the SEC23B gene, which encodes a core component of the coat protein complex II (COPII). F5F8D results from mutations in either LMAN1 (lectin mannose-binding protein 1) or MCFD2 (multiple coagulation factor deficiency protein 2), which encode the ER cargo receptor complex LMAN1-MCFD2. These diseases and their molecular pathogenesis are the focus of this review.

Absence of a Red Blood Cell Phenotype in Mice with Hematopoietic Deficiency of SEC23B

ABSTRACT Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.

Neural tube opening and abnormal extraembryonic membrane development in SEC23A deficient mice

COPII (coat protein complex-II) vesicles transport proteins from the endoplasmic reticulum (ER) to the Golgi. Higher eukaryotes have two or more paralogs of most COPII components. Here we characterize mice deficient for SEC23A and studied interactions of Sec23a null allele with the previously reported Sec23b null allele. SEC23A deficiency leads to mid-embryonic lethality associated with defective development of extraembryonic membranes and neural tube opening in midbrain. Secretion defects of multiple collagen types are observed in different connective tissues, suggesting that collagens are primarily transported in SEC23A-containing vesicles in these cells. Other extracellular matrix proteins, such as fibronectin, are not affected by SEC23A deficiency. Intracellular accumulation of unsecreted proteins leads to strong induction of the unfolded protein response in collagen-producing cells. No collagen secretion defects are observed in SEC23B deficient embryos. We report that E-cadherin is a cargo that accumulates in acini of SEC23B deficient pancreas and salivary glands. Compensatory increase of one paralog is observed in the absence of the second paralog. Haploinsufficiency of the remaining Sec23 paralog on top of homozygous inactivation of the first paralog leads to earlier lethality of embryos. Our results suggest that mammalian SEC23A and SEC23B transport overlapping yet distinct spectra of cargo in vivo.

Prediction of response and progression in multiple myeloma with serum free light chains assay: corroboration of the serum free light chain response definitions.

BACKGROUND The International Myeloma Working Group (IMWG) proposed response and progression criteria using serum free light chain (sFLC) testing for patients with nonsecretory multiple myeloma (MM). We attempt to validate these criteria by comparing paraprotein responses with sFLC responses in patients with secretory myeloma. PATIENTS AND METHODS Prospectively entered data for 89 patients with MM enrolled on various clinical trials at the Cleveland Clinic between April 2004 and December 2006 were reviewed. RESULTS By standard paraprotein criteria, 4 patients had complete remission (CR), 22 had partial remission (PR), 34 had stable disease (SD), 26 had progressive disease (PD), and 3 were inevaluable. Only 43 patients (48%) had an involved sFLC > or = 10 mg/dL (which is considered evaluable by the IMWG), of which 14 had PR, 8 had SD, 18 had PD, and 3 were inevaluable. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for sFLC in predicting response were 81%, 83%, 64%, and 92% respectively. The sensitivity, specificity, PPV, and NPV for sFLC in predicting progression were 93%, 80%, 72%, and 95% respectively. CONCLUSION sFLC reliably predicts response and progression in MM. However, half of the patients had inevaluable disease by sFLC, thus limiting the utility of sFLC testing in patients with nonmeasurable disease by electrophoretic methods.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice.

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.

Spontaneous 8bp deletion in Nbeal2 recapitulates the gray platelet syndrome in mice

During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10−7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.

Hematologic Manifestations of Deficiency of Adenosine Deaminase 2 (DADA2) and Response to Tumor Necrosis Factor Inhibition in DADA2-Associated Bone Marrow Failure

To the Editor, Deficiency of adenosine deaminase 2 (DADA2) was initially described simultaneously in 2014 by two groups [1, 2]. Enzymatic deficiency occurs via autosomal recessive inheritance of CECR1 gene mutations. Early descriptions of the disorder highlighted early-onset stroke, vasculitis presenting as polyarteritis nodosa, livedo reticularis, recurrent fevers, and hypogammaglobulinemia as the predominant clinical manifestations observed in patients [1–5]. The discovery of additional patients with DADA2 has revealed a highly variable clinical presentation with many features responsive to therapy with anti-tumor necrosis factor agents [2, 6–11]. Although not described in the initial cohorts of patients, hematological manifestations now appear to be a significant clinical component of deficiency of adenosine deaminase 2 [6–9, 12]. The pathogenesis underlying the hematologic dysfunction seen in patients has yet to be elucidated, although persistently elevated levels of tumor necrosis factor (TNF)-alpha have been described to be damaging to hematopoiesis within the bone marrow [13, 14]. Here, we describe a case of two adult brothers with DADA2 presenting with cytopenias and bone marrow hypocellularity. We particularly detail the clinical course of one brother with initial partial response to equine anti-thymocyte globulin and cyclosporine with recurrence of cytopenias following withdrawal of immunosuppression. Retreatment was initiated with anti-tumor necrosis factor alpha therapy resulting in clinical symptom improvement and normalization of blood counts. Patient 1 presented at 47 years of age with leukopenia following evaluation for recurrent upper respiratory and allergy symptoms. Complete blood count (CBC) at that time was notable for white blood cell (WBC) count 2.3 K/μL (35% neutrophils, 11% bands, 28% lymphocytes, 25% monocytes, and 1% basophils), hemoglobin 14.3 g/dL, MCV 90.7 fL, and platelets 153,000 K/μL. The patient’s past medical history was notable for Bmyositis^ as a child and right lower extremity cellulitis with associated sepsis as an adult. Family history was significant for a brother with neutropenia. Physical exam was unremarkable other than diffusely dry and vasculitic-appearing darkened skin on his bilateral lower extremities (Fig. 1a). Within 3 years, he progressed to pancytopenia with hemoglobin falling to 6 g/dL requiring frequent red blood cell transfusions and platelet and absolute neutrophil counts ultimately reaching nadirs of 49,000 and 620/uL, respectively. Initial bone marrow biopsy was hypocellular (range 0–15%) with a patchy distribution of hematopoietic precursors, mild reticulin fibrosis, and an increased proportion of CD3-positive T cells distributed interstitially. Flow cytometric immunophenotyping of the bone marrow disclosed no lymphoid antigen aberrancies and no increase in blasts. Cytogenetic analysis of the bone marrow yielded a normal male karyotype (46, XY). Testing for paroxysmal nocturnal hemoglobinuria was negative. Repeat bone marrow examination performed 6 months later, prior to * Thomas F. Michniacki tmich@med.umich.edu

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo

Significance In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type II, a disease of abnormal red blood cell development, while SEC23A deficiency results in cranio-lenticulo-sutural-dysplasia, a disease characterized by bone abnormalities due to defective collagen secretion (but no red blood cell defect). In this study, we show that SEC23A and SEC23B overlap in function, and that the disparate phenotypes of SEC23A/SEC23B deficiency within and across species are likely due to evolutionary shifts in gene-expression programs, rather than distinct functions of the SEC23 paralogs. Our studies provide a rationale for increased SEC23A or SEC23B expression as a therapeutic strategy for congenital dyserythropoietic anemia type II or cranio-lenticulo-sutural-dysplasia, respectively. Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.