Journal articles: 'CDAII'

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Bianchi, Paola, Klaus Schwarz, Elisa Fermo, Katja Heinrich, Cristina Vercellati, Anna Paola Maria Luisa Marcello, Richard van Wijk, et al. "Molecular Analysis of the SEC23B Gene In Patients Affected by Congenital Dyserythropoietic Anemia Type II (CDAII)." Blood 116, no. 21 (November 19, 2010): 4227. http://dx.doi.org/10.1182/blood.v116.21.4227.4227.

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Abstract Abstract 4227 CDAII, the most frequent type of congenital dyserythropoietic anemia, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblast morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Last year we and others identified SEC23B as the gene responsible for CDAII (Schwarz et al, 2009, Bianchi et al, 2009). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles from the endoplasmic reticulum to the Golgi apparatus. The gene, localized on chromosome 20p11, is split in 20 exons and codifies for a 767 aa protein. The aim of the study was to characterize the molecular defect in a large series of CDAII patients of Caucasian origin. Fifty-one CDAII patients from 49 unrelated families (17 Italians, 20 German or Swiss, 4 Dutch, 2 British, 2 Czech and 1 Greek, Turkish, Bulgarian and Irish origin) were analyzed by direct exon sequencing. We identified 36 different mutations, 25 of which were not described before. Eighteen were disruptive and 18 were missense mutations. The patients' molecular data are summarized in the Figure (novel mutations are reported in bold). All the missense mutations affected highly conserved aminoacids and were not found in 200 normal alleles examined. The c.325G>A mutation was identified in 15 homozygous patients and in two cases in combination with other mutations. The change c.40C>T was detected in 16 unrelated patients as heterozygous mutation and, for the first time, at the homozygous level in one patient only. Considering the entire series of patients (including previously published cases) characterized by our groups (86 CDAII patients from 77 unrelated families) c.325G>A and c.40C>T mutations account for 49% (76/154) of the unrelated mutated alleles and therefore should be firstly screened during molecular diagnosis of CDAII. A c.325G>A mutation usually results in a mild to moderate clinical picture at homozygous level, whereas it may cause a very severe clinical pictures when combined with other mutations (Fermo, et al 2010). Despite the sequencing of all exons and flanking intronic regions, 5 patients displayed only one mutation, suggesting the possibility that mutations could be located in regulatory regions or that a second gene could be involved in the pathogenesis of CDAII. P. Bianchi and K. Schwarz contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

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Barcellini, Wilma, Anna Ines Gregorini, Giulia Soverini, Anna Zaninoni, Juri A. Giannotta, Cristina Vercellati, Valeria Ferri, Paola Bianchi, Agostino Cortelezzi, and Maria Domenica Cappellini. "Iron Overload and Cytokine Serum Levels in Congenital Hemolytic Anemias." Blood 128, no. 22 (December 2, 2016): 2458. http://dx.doi.org/10.1182/blood.v128.22.2458.2458.

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Abstract Background: Congenital hemolytic anemias (CHAs) are a heterogeneous group of inherited RBC disorders including membrane and enzyme defects and dyserythropoietic anemias. Iron overload is a well recognized complication of hereditary hemoglobinopathies, both in transfusion-dependent and independent cases. However, little is known in congenital hemolytic anemias, with the exception of anecdotic reports in pyruvate kinase deficiency and dyserythropoietic anemias. Aim: to describe the clinical and hematological features at diagnosis and enrolment, to investigate iron overload by hepatic and cardiac T2* MRI, and to study inflammatory/regulatory cytokine profiles (IL-6, TNF-alpha, IFN-gamma, IL-10, IL-17) and hepcidin levels in patients with CHAs. Confounding factors such as hemocromatosis genotyping, metabolic syndrome, and hepatic viral profile were also considered. Methods: Between July 2015 and April 2016, 38 patients were enrolled (13 hereditary spherocytosis -HS, 3 hereditary stomatocytosis - HSt, 8 congenital dyserythropoietic anemia type II - CDAII, 13 pyruvate kinase deficiency - PKD, 1 glucose-phosphate isomerase deficiency). HS cases were enrolled on the basis of ferritin >300 ng/mL at diagnosis. Cytokine levels were detected in serum by ELISA. Comparisons were made by Students T test (continuous) and Fisher's exact test (categorical), and correlations by Pearson's linear coefficient. Results: The main clinical and hematological findings are shown in table. Median Hb values progressively decreased in the 4 groups considered, being close to normal in HS and moderately reduced in CDAII patients, whereas hemolytic parameters were comparable among groups. Consistently with clinical severity, ferritin values were particularly high in CDAII (together with transferrin saturation-TfS) and PKD patients, notwithstanding chelation in about half cases of both groups. Of note, only 2 PKD patients were transfusion-dependent, suggesting that other factors are involved in iron overload. Splenectomy had been performed in 17/38 (44.7%), mainly CDAII. Liver iron concentration (LIC) showed a great heterogeneity in all groups, with a trend towards higher values in CDAII; 16/36 (44%) patients had a LIC>4 mg/g DW (23%, 33%, 38% and 88% in HS, HSt, PKD and CDAII, respectively). Cardiac T2* value was normal in all subjects, with the exception of a HS and a CDAII case. Regarding possible cofactors, 12/16 displayed at least one of the following: 1 homozygous for HFE C282Y and 1 for H63D mutations, 3 HCV+, 4 BMI>25, 2 alcohol abuse, 3 heterozygous for HFE mutations. The following positive correlations were observed at enrolment: LIC and ferritin (r=0.68, p<0.05), LIC and TfS (r=0.34, p=0.05), and cardiac T2* and TfS (r=0.34, p<0.05). Moreover Hb values at diagnosis negatively correlated with LIC (r=0.37, p<0.05). Interestingly, among the 28 cases with ferritin <800 ng/mL, 10 (36%) displayed liver iron overload (LIC>4), of whom 5 with the above listed cofactors. As regards cytokine levels, IL-10 was significantly increased in HS, PKD and CDAII groups compared with normal cases; TNF-alpha was decreased in HS and PKD, and IFN-gamma increased in HS and CDAII. Ferritin values were positively correlated with IL-6 and IFN-gamma, and TfS negatively with IL-6 (r= -0.38, p<0.05). Hepcidin values were slightly increased in CHAs compared with normal controls, and correlated positively with ferritin (r=0.33; p<0.05), and negatively with TfS (r= -0.56; p<0.001). Finally, hepcidin levels were positively correlated with IL-6 (r=0.62; p<0.001), and negatively with IFN-gamma (r=0.3; p<0.05). Conclusion: Iron overload is highly prevalent in CHAs, particularly in PKD and CDAII, is independent from transfusion support, and is also observed in cases with ferritin <800 ng/mL. T2* MRI is the gold standard approach to evaluate iron overload in CHAs (as in other congenital anemias) and its use is advisable, particularly in the presence of cofactors, for early chelation. Cytokine studies suggest the existence of a positive loop among ferritin, hepcidin, and inflammatory cytokines such as IL-6 and IFN-gamma, and of a negative loop among TfS, hepcidin, and the same inflammatory cytokines. These findings disclose important hints to understand the multiple biological mechanisms of iron overload, and support the rationale for new emerging therapies. Table Table. Disclosures Barcellini: Agios: Consultancy.

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Khoriaty, Rami, Angela Weyand, Geoffrey Hesketh, Amélie Bernard, Lesley Everett, Guojing Zhu, Beth McGee, et al. "Overlap of SEC23A and SEC23B Function Suggests a Novel Therapeutic Approach for Congenital Dyserythropoietic Anemia Type II." Blood 130, Suppl_1 (December 7, 2017): 80. http://dx.doi.org/10.1182/blood.v130.suppl_1.80.80.

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Abstract 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. Disclosures No relevant conflicts of interest to declare.

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Khoriaty, Rami, Matthew P. Vasievich, and David Ginsburg. "The COPII pathway and hematologic disease." Blood 120, no. 1 (July 5, 2012): 31–38. http://dx.doi.org/10.1182/blood-2012-01-292086.

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Abstract 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.

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Khoriaty, Rami, Lesley Everett, Jennifer Chase, Guojing Zhu, Bin Zhang, Mark Hoenerhoff, Ivan Maillard, and David Ginsburg. "SEC23A Functionally Compensates for SEC23B Deficiency in Mice." Blood 126, no. 23 (December 3, 2015): 935. http://dx.doi.org/10.1182/blood.v126.23.935.935.

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Abstract 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. Disclosures No relevant conflicts of interest to declare.

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Russo, Roberta, Immacolata Andolfo, Luigia De Falco, Francesco Manna, Antonella Gambale, Mariasole Bruno, Gianluca De Rosa, Domenico Girelli, Lucia De Franceschi, and Achille Iolascon. "Erfe-Encoding FAM132B in Congenital Dyserythropoietic Anemia Type II." Blood 126, no. 23 (December 3, 2015): 535. http://dx.doi.org/10.1182/blood.v126.23.535.535.

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Abstract Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII), a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin (Iolascon, 2013). The most recently described hepcidin regulator is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-α superfamily that specifically inhibits hepcidin production in experimental models (Kautz, 2014). However, the function of ERFE in humans remains to be investigated. To determine whether dysregulation of ERFE expression is associated with ineffective erythropoiesis and iron-loading in CDAII, we studied the ERFE-encoding FAM132B gene expression in 48 SEC23B-related CDAII patients and 29 age and gender matched healthy controls (HCs). Twelve new cases and four novel SEC23B mutations were described. Samples were obtained after informed consent, according to the Declaration of Helsinki. Genomic DNA, mutational screening, RNA isolation, cDNA preparation, and qRT-PCR were performed as previously described (Russo, 2013). All patients were young adults (17.0±2.5 years at diagnosis), with increased serum ferritin (395.4±67.6 ng/mL) and transferrin saturation (71.9±5.4 %). We observed a statistically significant overexpression of FAM132B gene in peripheral blood mononuclear cells from CDAII patients (9.09±0.08) compared to HCs (8.32±0.12, p<0.0001). A similar trend was obtained when evaluating FAM132B expression in reticulocytes from a subset of patients and HCs. Of note, a statistically significant correlation between peripheral blood and reticulocyte FAM132B expression from the same patients was observed (Spearman ρ= 0.78, p=0.02). Although the role of ERFE in peripheral blood is still unknown, our observations suggested that the evaluation of FAM132B mRNA in peripheral blood is a reliable and easy-to-measure marker of ERFE levels. When we divided CDAII patients into two sub-groups accordingly to FAM132B gene expression, we observed a statistically significant reduction in hemoglobin (Hb) level in the high-FAM132B subset (8.6±0.4 g/dL) respect to low-FAM132B one (10.1±0.5 g/dL, p=0.02). Of note, the expression level of FAM132B did not correlate with the transfusion regimen. The higher amount of ERFE reflects the increased iron demand for Hb production as well as the expanding abnormal erythropoiesis, as attested by the increased RDW and sTfR (although not significant) in high-FAM132B patients. This in turn leads to reduced hepcidin in high-FAM132B group (4.2±1.8 nM) compared to low-FAM132B one (5.9±1.8 nM, p=0.05), resulting in augmented iron delivery to the erythron. Although the iron balance data do not differ significantly between the two groups, a tendency to decreased hepcidin/ferritin ratio and increased transferrin saturation was observed in high-FAM132B patients. Thus, FAM132B overexpression seems to contribute to the inappropriate suppression of hepcidin with subsequent hemosiderosis observed in CDAII. Consistent with our previous studies, we observed a reduced SEC23B expression in our patients compared to HC. Indeed, FAM132B and SEC23B gene expression exhibited an inverse correlation (Spearman ρ=-0.36, p=0.01). We confirmed the ex vivo data about inverse correlation between FAM132B and SEC23B expression observed in our patients by establishing K562 SEC23B-silenced cells. To knockdown SEC23B gene expression in K562 cells two different pGIPZ Lentiviral shRNAmir for SEC23B (shSEC23B-70/-74) were used. We observed a higher expression of FAM132B at 5 days of erythroid differentiation in K562 SEC23B-silenced cell compared to not-silenced ones. Conversely, SEC23B expression was lower in both shSEC23B compared to sh-CTR at 2 and 5 days of differentiation. Although the mechanisms of hemin-induced differentiation are quite different from EPO-induced ones, we can hypothesize that FAM132B over-expression is related to the maturative arrest and the subsequent increased number of erythroid precursors. This study provides the first analysis on ERFE regulation in humans. Our data suggest that ERFE over-expression in CDAII patients is the result of both physiological and pathological mechanisms leading to hepcidin suppression in condition of dyserythropoiesis. Nevertheless, it seems that ERFE cannot be the main erythroid regulator of hepcidin suppression, at least in CDAII patients. Disclosures No relevant conflicts of interest to declare.

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Khoriaty, Rami, Matthew Vasievich, Morgan Jones, Jennifer Chase, Lesley Everett, Bin Zhang, Ivan Maillard, and David Ginsburg. "SEC23B Deficiency Results in Different Phenotypes in Humans and Mice." Blood 124, no. 21 (December 6, 2014): 1341. http://dx.doi.org/10.1182/blood.v124.21.1341.1341.

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Abstract 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. Disclosures No relevant conflicts of interest to declare.

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Fermo, Elisa, Paola Bianchi, Cristina Vercellati, Wilma Barcellini, Carla Boschetti, Anna Paola Marcello, and Alberto Zanella. "Two Atypical Severe Cda Forms Presenting as Hydrops Foetalis Are Caused by Mutations in the SEC23B Gene." Blood 114, no. 22 (November 20, 2009): 1981. http://dx.doi.org/10.1182/blood.v114.22.1981.1981.

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Abstract Abstract 1981 Poster Board I-1003 Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of disorders characterized by ineffective erythropoiesis with prominent dysplastic features of the erythroid precursors in the bone marrow. Three main subtypes (I, II and III) have been identified, but several variants or atypical forms have been reported over the years. CDA are rarely associated with hydrops foetalis. In the past we described two cases presenting with hydrops foetalis and very severe anemia that were classified as atypical CDAs since they presented CDAII-like erythroblastic morphological features lacking other diagnostic CDAII markers. Very recently we and others (Bianchi et al, Human Mutat 2009, in press; Schwarz et al, Nat Genet 2009, in press) demonstrated that mutations in SEC23B gene, coding for a protein involved in the coat protein complex responsible for vesicle budding from the endoplasmic reticulum, cause CDA II. The aim of this work was to ascertain whether atypical CDAII-like forms presenting as hydrops foetalis could be caused by mutations in SEC23B gene and reclassified as CDAII. Two patients (Cantù-Rajnoldi et al, Br J Haematol 1997, 96: 530-3; Bianchi et al, Blood 1999, 94, suppl 1, 8b) from unrelated families with a history of intrauterine death of hydropic foetuses in previous pregnancies were referred at 20th week gestation following ultrasonic diagnosis of foetal hydrops and severe anemia (2.0 and 1.3 g/dL Hb respectively). In both cases intrauterine transfusions enabled the delivery of the babies. At birth both of them underwent extensive laboratory evaluation (including red cell metabolism and membrane proteins analysis) that was not informative on the causes of anemia. Ham test was repeatedly normal as for Western blotting analysis for GRP78 and glycoslylation pattern of red cell band 3. The bone marrow examination revealed the presence of 30 and 48% of bi- or multinucleated erythroblasts, some of whom presenting typical double outer membranes at transmission electron microscopy. Both babies became transfusion dependent. The 20 exons and intronic flanking regions of SEC23B gene were analyzed by direct sequencing. Both patients displayed mutations in SEC23B gene, in particular mutations c.325 G>A/ c.2101 C>T in patient 1 and c.325 G>A/ c.197G>A in patient 2 (Glu109Lys/ Arg701Cys and Glu109Lys/ Cys66Tyr respectively). c.325 G>A is the most frequent mutation so far described in CDAII and at homozygous level is usually associated with mild anemia. When found in compound heterozygosity with a second missense mutation as in these cases it may result in a very severe clinical pattern, although we cannot exclude that factors other than SEC23B mutations may contribute to worsening the clinical picture. In conclusion, SEC23B gene analysis allowed the correct classification of two very severe CDA cases associated with hydrops foetalis. This finding indicates that CDAII may result in intrauterine death and its frequency may therefore be underestimated. These cases may present as “atypical” for the lack of band3 hypoglycosylation likely due to the early sequestration of the more severely affected cells. Disclosures: No relevant conflicts of interest to declare.

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Khoriaty, Rami, Matthew Vasievich, Morgan Jones, Bin Zhang, Lesley Everett, Jiayi Tao, Ivan Maillard, and David Ginsburg. "Disparate Phenotypes Of SEC23B Deficiency In Humans and Mice." Blood 122, no. 21 (November 15, 2013): 312. http://dx.doi.org/10.1182/blood.v122.21.312.312.

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Abstract Congenital dyserythropoietic anemia type-II (CDAII) is an autosomal recessive disease characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from mutations in SEC23B, one of two closely related mammalian SEC23 paralogs. SEC23 is a core component of COPII coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum (ER) to the Golgi apparatus. Bone marrow transplantation cures CDAII, suggesting that the pathologic defect in this disease is restricted to the hematopoietic compartment. However, the mechanism by which SEC23B-deficiency results in CDAII remains unknown. We previously reported that mice homozygous deficient for SEC23B (Sec23bgt/gt) exhibit massive pancreatic degeneration. The latter resulted in perinatal mortality precluding evaluation of the adult hematopoietic compartment. To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, fetal liver cells (FLC) were harvested from Sec23bgt/gt or wildtype (WT) control E17.5 embryos and transplanted into lethally irradiated C57BL/6J mice. Recipients of Sec23bgt/gt FLC had normal peripheral blood counts and were indistinguishable from recipients of WT FLC. To test for a more subtle hematopoietic defect, Sec23bgt/gt FLCs were tested directly against WT FLC for their ability to reconstitute hematopoiesis in a competitive repopulation assay. SEC23B deficient FLC exhibited no competitive disadvantage at reconstituting erythropoiesis relative to WT FLC over 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. We also generated a second, conditional Sec23b allele, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 with Cre-recombinase results in a frame shift leading to a stop codon in exon 7. Mice with erythroid-specific SEC23B deficiency were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice maintained normal erythropoiesis indistinguishable from their WT littermates. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to p48-Cre or Pdx1-Cre transgenes generated phenotypes indistinguishable from complete SEC23B deficiency, demonstrating that loss of pancreatic Sec23b expression alone is sufficient to explain the perinatal lethality observed in Sec23bgt/gt and Sec23b-/- mice. Our results conclusively demonstrate that in contrast to humans, SEC23B-deficiency results in massive pancreatic degeneration in mice, but no CDAII in these animals. To investigate the cause of the disparate human and mouse SEC23B-deficient phenotypes, SEC23B/SEC23A expression ratios were examined in endogenous tissues from both species. This ratio (normalized to the ratio in liver mRNA as 1.0) was higher in mouse pancreas (12.7) compared to BM (2.6), with the reverse pattern observed in human BM (7.8) relative to pancreas (5.5). These data, taken together with the high degree of identity between SEC23A and SEC23B (∼ 85% amino acid identity), suggest that the tissue-specific functions of SEC23A and SEC23B may have shifted during evolution between humans and mice. To test the role of SEC23A, we generated a mouse with a conditional Sec23a allele, in which exon 3 is flanked by loxP sites (Sec23afl). Cre-recombinase mediated deletion of exon 3 results in a frame shift leading to a stop codon in exon 7 (Sec23a-). Mice with erythroid-specific SEC23A-deficiency (Sec23afl/-/EpoR-CreTg+) maintained normal red blood cell counts indistinguishable from their WT littermates. In summary, we have shown that SEC23B-deficient humans and mice exhibit disparate phenotypes. We have also demonstrated variations in the gene expression programs for SEC23A and SEC23B potentially explaining the pancreatic phenotype of SEC23B-deficiency in mice and the erythroid phenotype in humans. These results suggest that the two SEC23 paralogs have overlapping functions and that therapeutic strategies that increase the expression of either SEC23A or SEC23B in erythroid cells might be effective in CDAII. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII. Disclosures: No relevant conflicts of interest to declare.

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Villanueva, Melanie A., Carol J. Saunders, David Zwick, Laurie Smith, Darrell Dinwiddie, Emily Farrow, Neil Miller, Stephen Kingsmore, and Keith J. August. "An Unusual Presentation of Congenital Dyserythropoietic Anemia Type II (CDAII) Associated with Severe Anemia in a Patient with a Novel Mutation of the SEC23B Gene." Blood 120, no. 21 (November 16, 2012): 990. http://dx.doi.org/10.1182/blood.v120.21.990.990.

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Abstract Abstract 990 CDAII is an autosomal recessive disorder characterized by ineffective erythropoiesis, anemia, hemolysis, hepatosplenomegaly and morphologic abnormalities. While rare, more than 300 cases of CDAII have been described worldwide, making it the most common form of congenital dyserythropoietic anemia. In patients with CDAII, bone marrow specimens often demonstrate hypercellularity, erythroid hyperplasia and the presence of 10–45% bi- and multinucleated erythroid precursors. Generally, all patients will have a positive serum acid hemolysin test. The clinical picture includes a mild to moderate anemia and hemolysis with typical hemoglobin levels that range from 8 to 11 g/dL. In less than 10 percent of reported cases, severe anemia is seen requiring frequent red blood cell transfusions. Mutations in SEC23B are the underlying defect in the majority of patients with CDAII. SEC23B encodes an essential component of coat protein complex II coated vesicles, resulting in ineffective trafficking of secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Here we report a patient with a severe presentation of CDA II associated with a novel nonsense mutation in the SEC23Bgene. Our patient was born at 36 weeks estimated gestational age with IUGR and hypospadias. The family history included a brother born with severe anemia, severe hypospadias and hydrops fetalis who died at 4 days of life. Parents were of nonconsanguinious Samoan decent. The initial hemoglobin was 7 g/d; red blood cell transfusions were required on days of life 1, 10 and 15. Total bilirubin peaked at 3.3 and phototherapy was unnecessary. Now 20 months of age, he continues to be transfusion-dependent. Reticulocytopenia is pronounced with absolute reticulocyte counts consistently <20000/mL. Treatment with glucocorticoids produced a minimal increase in reticulocytes without a decrease in frequency of transfusions. Additional laboratory evaluation demonstrated mild indirect hyperbilirubinemia (1–3.4 mg/dL) that presented around one year of age, normal LDH and decreased haptoglobin. Bone marrow evaluation revealed dyserythropoiesis and reticulin fibrosis. Bi- and multinucleated cells were present and comprised 6% of the erythrocyte precursors. Exome sequencing was performed using Illumina hybrid capture, HiSeq sequencing and a full analysis pipeline. Sequencing revealed compound heterozygosity for two mutations in the SEC23B gene: c.53G>A (p.Arg18His) and c.1507C>T (p.Arg503X). The p.Arg18His has been previously reported in patients with CDAII. The p.Arg503X is a novel nonsense mutation that is expected to be pathogenic, likely resulting in nonsense-mediated decay of the mRNA. These mutations were confirmed clinically by Sanger sequencing and each parent was found to carry one mutation. Compound heterozygosity including a nonsense mutation in SEC23B has been reported to result in a more severe phenotype with reticulocytopenia, a finding consistent with the presentation of our patient. This case demonstrates that exome sequencing, with confirmatory dideoxy sequencing of the affected individual and the parents, can be a powerful new diagnostic approach in inherited hematologic disorders that feature genetic heterogeneity. Disclosures: No relevant conflicts of interest to declare.

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Fermo, Elisa, Paola Bianchi, Lucia Dora Notarangelo, Silvana Binda, Cristina Vercellati, Anna Paola Marcello, Carla Boschetti, Wilma Barcellini, and Alberto Zanella. "CDAII presenting as hydrops foetalis: Molecular characterization of two cases." Blood Cells, Molecules, and Diseases 45, no. 1 (June 2010): 20–22. http://dx.doi.org/10.1016/j.bcmd.2010.03.005.

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Bianchi, Paola, Elisa Fermo, Cristina Vercellati, Carla Boschetti, Wilma Barcellini, Alessandra Iurlo, Anna Paola Marcello, Pier Giorgio Righetti, and Alberto Zanella. "Identification of SEC23B as the Gene Responsible for Congenital Dyserythropoietic Anemia Type II using a Proteomic-Genomic Approach." Blood 114, no. 22 (November 20, 2009): 4028. http://dx.doi.org/10.1182/blood.v114.22.4028.4028.

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Abstract Abstract 4028 Poster Board III-964 CDAII, the most frequent type of congenital dyserythropoietic anemia family, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblasts' morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Recent studies indicated that CDAII is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts. Linkage analysis located the CDAII gene in a 5 cM region on chromosome 20 and several candidate genes have been excluded. We recently investigated the cytoplasmic proteome of human red blood cells (RBCs) using a combinatorial peptide ligand library as a capturing agent to amplify the signal of low- and very-low abundance proteins: 1578 proteins, most of them unexpected, were identified allowing a deep exploration of the RBC pathways (Roux-Dalvai, Mol Cell Proteomics, 2008). In this study we used a proteomic-genomic approach to identify the candidate gene for CDAII by matching the data on the cytoplasmic proteome of human RBC with the chromosomic localization of CDAN2 locus. The analysis of RBCs cytoplasmic proteome allowed us to identify 17 proteins codified by genes located in the chromosomic region between 20p11.23 and 20q11.23: SNX5, SEC23B, DTD1, NAT5, GINS1, BCL2L1, MAPRE1, CHMP4B, EIF2S2, AHCY, ACSS2, GSS, EIF6, CPNE1, EPB41L1, RPRD1B(C20orf77), TGM2. Most of them were excluded because found to be associated to other diseases, already excluded in CDAII by previous works, or because not related by function to CDAII. Among the remaining proteins we focus on SEC23B for its possible role in the endoplasmic reticulum-to Golgi trafficking and its localisation on 20p11, the region with the highest LOD-score in CDAII after the recent mapping of the markers on the current contigs (Denecke et al, Biochim Biophys Acta, 2009). The 20 exons and intronic flanking regions of SEC23B gene were analysed by direct sequencing in 16 CDA II patients from 13 families; 13 different mutations were detected among the 28 mutated alleles identified: 6 of them were missense, 2 frameshift, 1 splicing and 4 stop codon. All the missense mutations affected highly conserved aminoacids, and were not found in 100 normal alleles examined. Two of them (c.40C>T and c.325G>A) were detected in various unrelated patients. Despite all exons and flanking intronic regions were sequenced, one patient failed to show mutations and two cases displayed only one mutation, suggesting the possibility that a second gene could be involved in CDAII. Patients' data are summarised on the table (*,° = members of the same family). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles. Very recently it has been shown that knockdown of Zebrafish SEC23B leads to aberrant erythrocyte development (Schwarz et al, Nat Genet, 2009). Even if the exact function of human SEC23B is not completely clarified, abnormalities in this gene are likely to disturb ER-to Golgi trafficking affecting different glycosylation pathways, and ultimately accounting for the cellular phenotype observed in CDA II. Case Sex Origin Hb g/dL Retics 10e9/L Band3 hypoglyc Mutations Exons Effects 1 F N Italy 9.7 160 Yes c.40 C>T/c.428 A>CG 2/5 Arg 14 Trp/Frameshift 2 F C Italy 11.4 44 Yes c. 1821 delT/? 16/? Frameshift/? 3 F Bolivia 9.9 61 Yes c.568 C>T/c.1808 C>T 5/16 Arg 190 STOP/Ser 603 Leu 4 M Rumania 9.8 102 Yes c.40 C>T/c.1660 C>T 2/14 Arg 14 Trp/Arg 554 STOP 5 M S Italy 10.4 nd Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 6 F C Italy 8.3 103 Yes c.40 C>T/Ivs6 +1g>a 2/Ivs6 Arg 14 Trp/Splicing 7 M C Italy 9.7 100 Yes c.1489C>T/c. 2101 C>T 13/18 Arg 497 Cys/Arg 701 Cys 8° F N Italy 9.2 121 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 9° M N Italy 11.3 115 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 10° F N Italy 11.7 63 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 11 F N Italy 11.6 104 Yes c.40 C>T/c.1043 A>C 2/9 Arg 14 Trp/Asp 348 Ala 12* M S Italy 11.9 90 Yes c.40 C>T/c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 13* F S Italy 7.8 61 Yes c.40 C>T/ c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 14 F N Italy 9.5 105 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 15 M N Italy 10.4 236 Yes c.367 C>T/? 5/? Arg 123 STOP/? 16 F S Italy 10.7 87 Yes ?/? ?/? ?/? Disclosures: No relevant conflicts of interest to declare.

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Denecke, Jonas, and Thorsten Marquardt. "Congenital dyserythropoietic anemia type II (CDAII/HEMPAS): Where are we now?" Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1792, no. 9 (September 2009): 915–20. http://dx.doi.org/10.1016/j.bbadis.2008.12.005.

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Alloisio, N., P. Texier, L. Denoroy, C. Berger, E. Miraglia del Giudice, S. Perrotta, A. Iolascon, F. Gilsanz, G. Berger, and J. Guichard. "The cisternae decorating the red blood cell membrane in congenital dyserythropoietic anemia (type II) originate from the endoplasmic reticulum." Blood 87, no. 10 (May 15, 1996): 4433–39. http://dx.doi.org/10.1182/blood.v87.10.4433.bloodjournal87104433.

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We studied 20 individuals from 17 unrelated families with congenital dyserythropoietic anemia (type II; CDAII). The clinical phenotype was mild to moderate. The inheritance pattern was invariably recessive. Coomassie blue stained gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) show that band 3 was thinner and migrated slightly faster than usual. In addition, staining showed two unknown minor bands (in the patients), but not in normal controls, the obligate carrier parents, or in patients with other anemic syndromes. These minor proteins were studied using partial digestion, amino acid sequencing, Western blotting, immunofluorescence, and immunogold electron microscopy. They were identified as the glucose-regulated protein GRP78 and calreticulin that are resident proteins of the endoplasmic reticulum (ER). Using specific antibody, we showed that protein disulfide isomerase (PDI), a third major protein of the ER, was also present on the SDS-PAGE of red blood cell (RBC) ghosts. Immunofluorescence colocalized PDI with the dense discontinuous ring decorating the RBC membrane. Immunogold electron microscopy showed that PDI was localized in the lumen of the cisternae, confirming that these originate from the smooth ER. From a practical point of view, screening the above minor proteins in RBC membranes appears to be a straightforward and reliable diagnostic test for CDAII.

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King, Richard, Ann Friedman, Zesen Lin, and Rami Khoriaty. "Functional Overlap between the SEC23 Paralogs Suggests a Novel Treatment Paradigm for Congenital Dyserythropoietic Anemia Type II." Blood 134, Supplement_1 (November 13, 2019): 2221. http://dx.doi.org/10.1182/blood-2019-129669.

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Congenital dyserythropoietic anemia type II (CDAII), an autosomal recessive disease characterized by ineffective erythropoiesis and increased percentage of bi-nucleated erythroid precursors in the bone marrow (BM), results from loss of function mutations in SEC23B, which encodes a core component of COPII vesicles. Approximately 8,000 secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII vesicles, suggesting that a defect in this pathway would result in a profound systemic phenotype. However, CDAII patients exhibit a specific erythroid phenotype, with no other defects described. Mammals have 2 paralogs for SEC23, SEC23A and SEC23B. In contrast to SEC23B mutations, bi-allelic SEC23A loss of function mutations in humans result in cranio-lenticulo-sutural dysplasia, a disease characterized by skeletal defect but normal erythropoiesis. We previously demonstrated that a SEC23B-A chimeric protein composed of the first 122 amino acids of SEC23B followed by amino acids 123-765 of SEC23A overlaps in function with SEC23B, suggesting that the 2 SEC23 paralogs are functionally interchangeable. However, to rule out the possibility that the functional overlap was due to the first 122 amino acids of SEC23B, we generated a bacterial artificial chromosome (BAC) transgene that expresses the full Sec23a coding sequence from the endogenous genomic locus of Sec23b (Sec23b-a BAC). We crossed the Sec23b-a BAC to the Sec23b null allele (Sec23b-) and demonstrated that this BAC rescues the phenotype of mice deficient in Sec23b (Sec23b-/-). Therefore, we now conclusively demonstrate that the SEC23A protein functionally replaces SEC23B when expressed from the endogenous regulatory elements of Sec23b. We have previously shown that mice with erythroid-specific and pan-hematopoietic SEC23B deficiency exhibit a normal erythroid phenotype. In light of the functional overlap between SEC23A and SEC23B, we hypothesized that mice with erythroid-specific deficiency for SEC23A, alone or in combination with SEC23B, might exhibit an erythroid phenotype. First, we generated mice with erythroid-specific (EpoR-Cre) SEC23A deficiency. These mice were observed at the expected Mendelian ratios at weaning. Complete (or near complete) excision of the Sec23a floxed (Sec23afl) allele was confirmed in the erythroid cells. Peripheral blood counts, BM cellularity and morphology, and percent and distribution of BM erythroid cells among the 5 stages of maturation were indistinguishable between mice with erythroid SEC23A deficiency and wildtype littermate controls. Additionally, the percentage of bi-nucleated erythroid precursors were not increased in Sec23afl/flEpoR-Cre+ mice. Thus, mice with erythroid-specific SEC23A deficiency do not exhibit an erythroid phenotype. Similarly, mice with pan-hematopoietic SEC23A deficiency (Vav1-Cre) do not exhibit a hematologic phenotype. Next, we generated mice with Sec23a deletion and Sec23b haploinsufficiency in the erythroid compartments. These mice exhibited normal survival, a mild reduction in hemoglobin levels (p = 0.014), and a block in late erythroid maturation (Stage V erythroid cells were reduced to 22.6% compared to 30.3% in control mice; p=0.08). In contrast, mice with erythroid-specific deletion for all 4 Sec23 alleles (combined SEC23A/B deficiency) died at mid-embryogenesis exhibiting reduced size and appearing pale compared to wildtype littermate controls, with histologic evidence of dyserythropoiesis reminiscent of human CDAII. Overall, these results suggest a requirement for a threshold level of total SEC23 (combined SEC23A/B) expression in the erythroid compartment. These results also suggest that the defect in CDAII is intrinsic to the RBC. Finally, we generated K562 cells with either SEC23B or SEC23A deletion using CRISPR/Cas9 genome editing. SEC23B or SEC23A deletion alone was tolerated in the K562 cells. However, combined deletion of SEC23A and SEC23B was not tolerated. Taken together, the results summarized above demonstrate that SEC23A and SEC23B appear to compensate for one another's function in murine and human erythroid cells. This finding suggests a potential therapeutic role for increasing expression of SEC23A to compensate for SEC23B deficiency in CDAII. This work is currently ongoing. Disclosures No relevant conflicts of interest to declare.

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Esposito, Maria Rosaria, Roberta Russo, Annaelena Troiano, Immacolata Andolfo, Roberta Asci, and Achille Iolascon. "In Vitro Characterization of R14W Mutation in SEC23B, the CDAII Causative Gene." Blood 118, no. 21 (November 18, 2011): 2098. http://dx.doi.org/10.1182/blood.v118.21.2098.2098.

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Abstract Abstract 2098 Congenital dyserythropoietic anemias (CDAs) designate a group of genetic disorders in which ineffective erythropoiesis is the predominant mechanism of anemia marked by distinct morphologic abnormalities of the majority of erythroblasts in the bone marrow. CDA type II (CDAII) is the most common type of CDA. It is characterized by a recessive model of inheritance, mild to moderate anemia, jaundice, and splenomegaly (Fukuda MN, Glycobiology 1990; Fukuda MN, Clin Haematol 1993), by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance (Schwarz K and Iolascon A. et al., Nat Genet. 2009). The specific hallmark of diagnosis is the presence of the more abundant protein of membrane red cell, band 3, in a hypoglycosilated state; this is thinner and migrated slightly faster than in controls on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Anselstetter V et al., Br J Haematol 1977). The causative gene of CDAII, SEC23B, is a member of the SEC23 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec23p (66.4%) component of COPII, the coat protein complex responsible for vesicle budding from the ER. The function of this gene product has been implicated in cargo selection and concentration. The SEC23B gene spans approximately 54 kb on human chromosome 20p11.23 and codifies for a protein of 767 aminoacids divided in five functional domains: zink finger, trunk, β-sheet, helical and gelsolin domain. Although most of the SEC23B mutations are sporadic events, 4 mutations (R14W, E109K, R497C, I318T) accounted for more than 50% of mutated alleles. The aim of this study is the in vitro characterization of the R14W mutation, the most frequent variant in Italy, particularly in South of Italy (Russo R et al., Am J Hematol. in press). We used the human erythroleukemia cell line, HEL, as in vitro model because it is more similar to mature red cell. By using in silico tool ESyPred3D Web Server 1.0, we predicted that this aminoacidic substitution alters the zink finger domain 3D structure, when compared to wild type protein. This tool implements homology modeling approach followed by a final analysis with MODELLER release 4 in order to build a 3D model of the submitted protein (Lambert et al, 2002). However, when we transfected the SEC23B-R14W we observed a strong reduction of gene expression in the mutant when compared to SEC23B-wt construct by qRT-PCR. These results have been also confirmed at the protein level. In fact the protein expression of SEC23B-R14W showed a reduction comparable to gene expression respect to SEC23B-wt construct. Immunofluorescence analyses by confocal microscopy, were used for the investigation of the cellular localization of SEC23B-R1W protein and, interestingly, the localization of mutant protein was not changed when compared to that wt. Our data allow us to hypothesize that the mutation R14W gives rise to anomalous protein product quantity, but the protein function would be like not altered. Our findings demonstrated that the most frequent mutation found in Italy, SEC23B-R14W, results in a reduced half-life of the mutated mRNA, without altering the cellular localization in HEL cell line. SEC23B belongs to a multiproteic compelx that assembles with the others complex proteins in accordance with a specific structure. Each structure establishes a cargo selectivity. Further studies are necessary in order to understand what is the role of SEC23B in selectivity of the cargo in erythroid cells and how its disruption could determines the appearance of the principal pathological phenotype in CDAII patients, for example the hypoglycosilation of band 3. Disclosures: No relevant conflicts of interest to declare.

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Russo, Roberta, Maria Rosaria Esposito, Daniela Spano, Luigia De Falco, Roberta Asci, Carmelo Piscopo, Immacolata Andolfo, and Achille Iolascon. "COPII Complex Characterization During Erythroid Differentiation and Its Involvement in CDAII Disease." Blood 114, no. 22 (November 20, 2009): 3009. http://dx.doi.org/10.1182/blood.v114.22.3009.3009.

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Abstract Abstract 3009 Poster Board II-985 Congenital dyserythropoietic anemia type II (CDA II) is an autosomal recessive disorder affecting the normal differentiation-proliferation pathway of the erythroid lineage. It comprises an anemia of variable severity, jaundice, and variable splenomegaly. Erythroid hyperplasia with binuclearity or multinuclearity involving late erythroblasts in the bone marrow (BM) is a key feature of the diagnosis (Iolascon, 2001). In addition, on electron microscopy, vesicles of endoplasmic reticulum (ER) appear to be running beneath the plasma membrane (Alloisio, 1996). The principal biochemical feature is the hypoglycosilation of several proteins, such as transferrin and band 3 (Anselstetter, 1977). Recently, we identified SEC23B as the CDA II causative gene (Schwarz, 2009). The SEC23B gene encodes the SEC23B component which is part of the cytoplasmic coat protein (COP)II complex. COPII coated vesicles bud from the endoplasmic reticulum to export newly synthesized proteins to the trans Golgi. In yeast, COPII coated vesicles form by the sequential binding of Sar1-GTP, the inner complex proteins Sec23-Sec24 and the outer complex components Sec13-Sec31 on the endoplasmic reticulum (ER) (Fromme, 2008). Our aim was to characterize the COPII complex in CD34+ progenitor cells during erythroid differentiation by gene expression analysis. Mononuclear cells from the peripheral blood of normal subjects were isolated on a Ficoll-Hypaque gradient and CD34+ progenitors were separated on immuno-affinity columns. For erythroid differentiation, CD34+ cells of three different pools were separately plated on plastic culture dishes in methylcellulose medium containing 3U/mL erythropoietin (EPO) (Pinho, 2008). The cells have been cultured for 7 and 14 days after EPO treatment. Quantitative real time (qRT)-PCR on CD34+ during erythroid differentiation was performed to assess the gene expression level. The relative gene expression was calculated by 2*(-ΔCt) method (Livak, 2001), using GAPDH gene as internal control (figure 1). Mammalian orthologues have been identified for each of the five proteins involved in COPII coat formation. In humans, two isoforms of Sec23, Sar1 and Sec31 and four mammalian isoforms of Sec24 have been reported (Kuge, 1994; Paccaud, 1996; Wendeler, 2007; Mancias, 2008; Shugrue, 1999; Tang, 2000; Stankewich, 2005). We already demonstrated that during normal erythropoiesis a SEC23A down regulation is associated to SEC23B upregulation. These data suggest that SEC23B mutants could disrupt the COPII complex in erythroid lineage, and consequently induce CDA II (Schwarz, 2009). On the contrary, the two isoforms of SAR1 showed the same trend during erythroid differentiation: however, SAR1A isoforms has an higher expression when compared to SAR1B isoform. Among the four SEC24 isoforms, SEC24A, B and C showed the same increased expression after EPO treatment; SEC24D, indeed, showed a decreasing trend during differentiation time. Only one form of mammalian SEC13 has been described (Shaywitz, 1995), and it showed an increased expression after EPO treatment. Between SEC31 mammalian isoforms, SEC31A showed overall an higher gene expression compared to SEC31B gene. The gene expression analysis of SEC12, the transmembrane guanine nucleotide exchange factor that catalyzes the COPII vesicle formation by GDP-GTP exchange on Sar1 (Sato, 2004), revealed an higher mRNA level at 14 days when compared to 7 days after EPO treatment. Here, we have identified the isoforms of COPII complex most expressed during erythroid differentiation. This study could allow us to clarify the role of each COPII gene in erythroid lineage, and to identify other genes potentially involved in CDA II pathogenesis.Figure 1.Gene expression profile during CD34+ erythroid differentiation. Data are presented as mean ± standard deviation. *p value < 0.05 calculated on day 0 by Student t test corrected by Bonferroni method.Figure 1. Gene expression profile during CD34+ erythroid differentiation. Data are presented as mean ± standard deviation. *p value < 0.05 calculated on day 0 by Student t test corrected by Bonferroni method. Disclosures: No relevant conflicts of interest to declare.

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Bianchi, Paola, Elisa Fermo, Cristina Vercellati, Carla Boschetti, Wilma Barcellini, Alessandra Iurlo, Anna Paola Marcello, Pier Giorgio Righetti, and Alberto Zanella. "Congenital dyserythropoietic anemia type II (CDAII) is caused by mutations in theSEC23Bgene." Human Mutation 30, no. 9 (September 2009): 1292–98. http://dx.doi.org/10.1002/humu.21077.

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Khoriaty, Rami, Geoffrey G. Hesketh, Amélie Bernard, Angela C. Weyand, Dattatreya Mellacheruvu, Guojing Zhu, Mark J. Hoenerhoff, et al. "Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo." Proceedings of the National Academy of Sciences 115, no. 33 (July 31, 2018): E7748—E7757. http://dx.doi.org/10.1073/pnas.1805784115.

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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.

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Dessy-Rodriguez, Mercedes, Sara Fañanas-Baquero, Veronica Venturi, Salvador Payán-Pernía, Cristian Tornador, Gonzalo Hernandez, Mayka Sanchez, José C. Segovia, and Oscar Quintana Bustamante. "Modelling Congenital Dyserythropoietic Anemia Type II through Gene Editing in Hematopoietic Stem and Progenitor Cells." Blood 136, Supplement 1 (November 5, 2020): 27. http://dx.doi.org/10.1182/blood-2020-139207.

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Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

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Zaninoni, Anna, Roberta Russo, Roberta Marra, Elisa Fermo, Immacolata Andolfo, Anna Paola Marcello, Dario Consonni, et al. "Evaluation of the Main Regulators of Systemic Iron Homeostasis in Pyruvate Kinase Deficiency." Blood 138, Supplement 1 (November 5, 2021): 1993. http://dx.doi.org/10.1182/blood-2021-151635.

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Abstract Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias (CDA), and some forms of congenital hemolytic anemias. Among them pyruvate kinase deficiency (PKD) has been shown to develop iron overload also in absence of transfusions suggesting dyserythropoietic features. Moreover, severe forms can be misdiagnosed as CDA due to bone marrow abnormalities and ineffective erythropoiesis further supporting this evidences. The hormone erythroferrone (hERFE) is produced by erythroblasts in response to erythropoietin (EPO), and acts by suppressing hepcidin, thereby increasing iron absorption and mobilisation for erythropoiesis demand. The ERFE-hepcidin axis seems to play a crucial role in the pathogenesis of these disorders; an increased erythroferrone release by immature erythroid cells results in hepcidin suppression and secondary iron overload that could finally results in ineffective erythropoiesis and anemia. To investigate the pathophysiological basis of iron overload in PKD, we analysed the levels of hERFE, EPO, hepcidin, and soluble transferrin receptor (sTFR) in a large group of 41 PKD patients equally distributed by gender, age and severity. The results were analysed in comparison with two groups of patients affected by hemolytic anemia with overt dyserythropoiesis (42 patients with CDA type II) and with congenital hemolytic anemia due to RBC membrane defects (51 patients with hereditary spherocytosis [HS]), respectively. Demographic, hematologic, and biochemical features of the three groups of patients are reported in the table. Among the PKD patients, 18/41 were <18 yrs, median Hb level at the time of the study was 9.05g/dL (range 5.5-14.5), 12 underwent splenectomy, 28 ever received at least three transfusions their life, 14 of them transfusion dependent (>6 tx/yrs). Mean ferritin levels at the time of the study were 546 ng/ml (range 59-4990), 15/41 patients requiring chelation therapy for iron overload developed also in absence of transfusions. As expected, CDAII patients showed decreased hepcidin levels (3.74 ng/mL; n.v. 17.25, P<0.001) associated with increased erythropoietin (62.7 IU/L, n.v. 6.5, P=0.01) and hERFE (24.8 ng/mL, n.v. 1, P<0.0001). On the contrary, HS showed increased hepcidin, with less marked increased of ERFE (9.9 ng/mL, P=0.02) and EPO (36.4IU/L, P=0.005). In PKD patients we observed decreased hepcidin levels (7.15 ng/mL, P=0.03)), increased hERFE (18ng/mL, P<0.0001) and EPO (75.6 IU/L, P=0.009). Instead, sTFR was equally increased in the three groups of patients (Figure). Interestingly, by comparing the three groups of patients, PKD showed dyserythropoietic features as evidenced by the observation of intermediate values between HS and CDAII of hepcidin (P=0.007 PKD v CDAII and P=0.0002 PKD vs HS), hEFRE, and sTFR. This study provides the first analysis of the main regulators of systemic iron homeostasis in PK deficiency compared either with the model of a structural RBC defect (HS) or with the typical model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. These data provide evidence of the dyserythropoietic features of PK deficiency, underlining the need of accurate diagnosis and paving the way of novel therapeutic approaches in PK deficiency. Zaninoni A. and Russo R. equally contributed to the study Figure 1 Figure 1. Disclosures Fattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Annexon: Consultancy; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Barcellini: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria. Iolascon: Bluebird Bio: Other: Advisory Board; Celgene: Other: Advisory Board. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Swenson, Anna, Katherine Womack, Meaghan Quinn, and Gary Curhan. "METHODS TO DERIVE A CROHN'S DISEASE ACTIVITY INDEX FROM ELECTRONIC HEALTH RECORDS DATA." Inflammatory Bowel Diseases 29, Supplement_1 (January 26, 2023): S20—S21. http://dx.doi.org/10.1093/ibd/izac247.039.

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Abstract BACKGROUND The Crohn’s Disease Activity Index (CDAI) is currently the gold-standard for measuring disease activity in Crohn’s disease (CD) patients. It is the most commonly used outcome measure in clinical trials, however, due to the burden of completion, it is infrequently used in routine care. Real world data, including electronic health records (EHR), are useful for studying longitudinal disease and treatment outcomes in the real world, but most EHR databases do not include CDAI. Building off the methods presented by Rudrapatna et al. 2021, we calculated a derived CDAI using data elements available in EHRs. METHODS Data were derived from the OM1 PremiOM UC Dataset (OM1, Boston, MA), a multisource real-world database with linked healthcare claims and EHR data on US patients with CD (2013-present). Weight, body temperature and hematocrit were sourced from structured vital signs and laboratory data. Ideal body weight was calculated from height and gender using the Devine formula. Extraintestinal manifestations/complications were evaluated based on the presence or absence of ICD diagnosis codes for each condition. Abdominal mass was abstracted from clinical notes. Number of stools in the past 7 days, average daily abdominal pain, and general well being were extrapolated from single-day measures extracted from clinical notes rather than based on 7-day diaries. Each component value was multiplied by the weights outlined in Best et al. 1976. Components recorded within 4 months were added to calculate a derived CDAI. Multiple CDAIs could be calculated for each patient over time, but each component was allowed to contribute to only one CDAI score. RESULTS Among 13,159 CD patients, only 12 had a CDAI recorded in their notes. After applying the algorithm, we were able to calculate a derived CDAI for 1,378 patients. 489 patients had > 1 score derived. Figure 1 shows the distribution of derived CDAIs. Patients with a derived CDAI were younger and a higher proportion were treated with biologics and corticosteroids than those who could not have a CDAI calculated (Table 1). CONCLUSIONS A derived CDAI can be calculated from data that may be routinely recorded in EHRs. Efforts to standardize the collection of CDAI components in EHRs would further increase the availability of derived CDAIs. Further work to validate these derived scores against a gold standard and to replicate the findings in other real-world datasets should be considered. Validation was not feasible in this dataset due to the lack of recorded CDAIs. Derived CDAIs could potentially be used to study longitudinal changes in disease status and treatment effectiveness in settings where observed CDAIs are unavailable.

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Vasievich, Matthew, Bin Zhang, Jesse Rinehart, Morgan Jones, Ivan Maillard, and David Ginsburg. "Sec23b deficiency In Mice Results In Pancreatic Destruction and Defective long Term Hematopoietic Stem Cell Function." Blood 116, no. 21 (November 19, 2010): 2038. http://dx.doi.org/10.1182/blood.v116.21.2038.2038.

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Abstract Abstract 2038 Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disorder caused by mutations in the gene SEC23B. SEC23B is a component of the COPII coat complex that transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. CDAII is characterized by mild to moderate anemia and >10% bi- or multi- nucleate erythroblasts in the bone marrow. We generated Sec23b deficient mice (Sec23b gt/gt) from ES cells with a genetrap cassette inserted into the last intron of Sec23b. Sec23b gt/gt mice die at birth with destruction of the exocrine pancreas. Eight to 12 week old lethally irradiated C57BL/6J recipients were transplanted with 106 E17.5 fetal liver cells from either wildtype or Sec23b gt/gt mice. Mice were bled at 6, 8 and 12 weeks post transplant and no significant difference was observed in either hematocrit or hemoglobin (N=3-7 mice per group). Red blood cell (rbc) ghosts prepared from peripheral blood of transplanted mice showed no shift of Band 3 on SDS-PAGE analysis. Ghost fractions from Sec23b gt/gt recipients analyzed by western blot for the presence of residual ER proteins showed no difference between wildtype and Sec23b gt/gt fetal liver cell recipients. No significant difference was found in bi-nucleate erythroblasts in the bone marrow, myeloid:erythroid ratio or spleen mass. We quantitatively analyzed the proteome of mature rbc ghosts from Sec23b gt/gt recipients by Stabile Isotope Labeling of Amino acids in Cells (SILAC) proteomics using rbc ghosts from mice fed lysine labeled with 13C (SILAC chow). When SILAC-labeled rbc ghosts were compared by mass spectrometry analysis with ghosts from Sec23b gt/gt fetal liver cell recipients, no significant differences in abundance of rbc membrane proteins were observed. To more stringently test the ability of Sec23b gt/gt fetal liver cells to engraft, 5×105 wildtype or Sec23b gt/gt fetal liver cells were co-transplanted with 5×105 wildtype fetal liver cells expressing a GFP transgene. FACS analysis of peripheral blood obtained at 6 and 8 weeks post transplant showed significant out-competition of Sec23b gt/gt cells by GFP+ progenitors in Ter119+ rbcs (p<0.01, at all time points), CD3+ T cells (p<0.01 at 8 weeks), B220+ B cells (p<0.01, at all time points) and Mac1+Gr1+ myeloid cells (p<0.01, at all time points) compared to wildtype controls (N=5-7 mice per group). Fetal livers from wildtype and Sec23b gt/gt mice showed no difference in the total number of CD150+CD48-Sca1+cKit+(lineage-) long term hematopoietic stem cells (LT-HSCs) per fetal liver (p=0.45). In conclusion, Sec23b deficient humans and mice exhibit disparate phenotypes, apparently restricted to CDAII in humans and a prominent neonatal pancreatic insufficiency in mice. These differences could be due to evolutionary changes in relative expression and/or function of the Sec23a and b paralogs in humans and mice. However, we cannot exclude an allele-specific defect due to residual or aberrant function of the Sec23b gt allele. Analyses of a deleted and a conditional floxed Sec23b null allele are currently in progress. Finally our transplant data suggest that Sec23b traffics cargoes important for the function of murine LT-HSCs. This finding contrasts with the apparently erythroid-specific human phenotype, raising the possibility of a requirement for Sec23b function in the marrow microenvironment not tested by these transplant studies. Tissue specific deletion of Sec23b is currently in progress to address this question. Disclosures: No relevant conflicts of interest to declare.

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Bianchi, Paola, Klaus Schwarz, Josef Högel, Elisa Fermo, Cristina Vercellati, Regine Grosse, Richard van Wijk, et al. "Analysis of a cohort of 101 CDAII patients: description of 24 new molecular variants and genotype-phenotype correlations." British Journal of Haematology 175, no. 4 (July 29, 2016): 696–704. http://dx.doi.org/10.1111/bjh.14271.

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Pellegrin, Stéphanie, Katy L. Haydn-Smith, Lea A. Hampton-O'Neil, Bethan R. Hawley, Kate J. Heesom, Elisa Fermo, Paola Bianchi, and Ashley M. Toye. "Transduction with BBF2H7/CREB3L2 upregulates SEC23A protein in erythroblasts and partially corrects the hypo-glycosylation phenotype associated with CDAII." British Journal of Haematology 184, no. 5 (March 14, 2018): 876–81. http://dx.doi.org/10.1111/bjh.15189.

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Dessy-Rodriguez, Mercedes, Sara Fañanas-Baquero, Veronica Venturi, Salvador Payan, Cristian Tornador, Gonzalo Hernández, Paola Bianchi, et al. "Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II." Blood 138, Supplement 1 (November 5, 2021): 1994. http://dx.doi.org/10.1182/blood-2021-152332.

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Abstract Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients. Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction. In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. Disclosures Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

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Khoriaty, Rami, Matthew Vasievich, Morgan Jones, Lesley Everett, Bin Zhang, Ivan Maillard, and David Ginsburg. "Disparate SEC23B Deficient Phenotypes in Humans and Mice." Blood 120, no. 21 (November 16, 2012): 974. http://dx.doi.org/10.1182/blood.v120.21.974.974.

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Abstract Abstract 974 SEC23 is a core component of the COPII coated vesicle that mediates the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. Mutations in human SEC23B, one of two mammalian SEC23 paralogs, results in the autosomal recessive disease, congenital dyserythropoietic anemia type II (CDAII). In contrast, SEC23B deficient mice were recently reported to die perinatally with massive pancreatic degeneration, with no evidence of anemia at birth (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9.) To examine the impact of SEC23B deficiency on hematopoietic function in mice surviving beyond the immediate perinatal period, lethally irradiated C57BL/6J mice were transplanted with fetal liver cells (FLC) collected from E17.5 embryos that were either wild-type (WT) or homozygous for the previously reported Sec23b gene trap allele (Sec23bgt/gt). Recipients of Sec23bgt/gt FLC were indistinguishable from their WT transplant controls and exhibited none of the phenotypic characteristics of CDAII (anemia, increased bi/multi-nucleated RBC precursors, narrower band size and increased shift of the membrane protein band 3 on SDS gel electrophoresis, or RBC double membrane appearance by electron microscopy). To test for a more subtle defect in hematopoietic reconstitution, Sec23bgt/gt FLCs were co-transplanted with WT GFP+ FLC in a 1:1 ratio, with no competitive difference observed over the course of 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. To rule out an incidental mutation in a nearby gene (“passenger gene”) as the cause of the pancreatic phenotype in SEC23B-deficient mice, two Sec23b bacterial artificial chromosome transgenes were crossed into the Sec23bgt line, with both demonstrating complete rescue of the Sec23bgt/gt pancreatic phenotype, with normal survival to adulthood. Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein consistent with the gene trap insertion into Sec23b intron 19, and this fusion protein co-immunoprecipitates with SEC24A (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9). To rule out a contribution of the SEC23B/βGEO fusion protein to the disparate human and mouse SEC23B-deficient phenotypes, a second, conditional SEC23B allele was analyzed, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 results in frame shift and stop codon in exon 7. Mice with an erythroid-specific deficiency of SEC23B were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice exhibit no anemia compared to their WT litter mates. Transplant recipients of FLCs from E16.5 embryos homozygous for a germline deletion of Sec23b exons 5 and 6 (Sec23b−/−) were also indistinguishable from mice receiving WT FLCs. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre transgene confirmed the pancreatic phenotype observed. This suggests that the perinatal lethality in Sec23b deficient mice may be the result of the loss of pancreatic Sec23b expression. To explore the mechanism for the disparate human and mouse SEC23B-deficient phenotypes, the expression patterns for SEC23A and SEC23B were examined in mice and humans by RT PCR and western blotting. The ratio of SEC23B/SEC23A expression is higher in mouse pancreas (12.7) compared to bone marrow (2.6), whereas in humans, the ratio is higher in the bone marrow (7.8) relative to pancreas (5.5) (normalized to liver). Taken together with the high degree of sequence similarity between SEC23A and SEC23B (∼ 85% identity at the amino acid level), we hypothesize that these 2 SEC23 paralogs overlap extensively at the level of protein function, with the disparate deficiency phenotypes due primarily to differences in tissue and developmentally-specific gene expression programs that have shifted extensively during recent mammalian evolution. These findings have important implications for the comparative function of other closely related paralogous genes. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII and potential therapeutic approaches. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

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Iolascon, Achille, Immacolata Andolfo, and Roberta Russo. "Congenital dyserythropoietic anemias." Blood 136, no. 11 (September 10, 2020): 1274–83. http://dx.doi.org/10.1182/blood.2019000948.

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Abstract Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of inherited anemias that affect the normal differentiation–proliferation pathways of the erythroid lineage. They belong to the wide group of ineffective erythropoiesis conditions that mainly result in monolinear cytopenia. CDAs are classified into the 3 major types (I, II, III), plus the transcription factor-related CDAs, and the CDA variants, on the basis of the distinctive morphological, clinical, and genetic features. Next-generation sequencing has revolutionized the field of diagnosis of and research into CDAs, with reduced time to diagnosis, and ameliorated differential diagnosis in terms of identification of new causative/modifier genes and polygenic conditions. The main improvements regarding CDAs have been in the study of iron metabolism in CDAII. The erythroblast-derived hormone erythroferrone specifically inhibits hepcidin production, and its role in the mediation of hepatic iron overload has been dissected out. We discuss here the most recent advances in this field regarding the molecular genetics and pathogenic mechanisms of CDAs, through an analysis of the clinical and molecular classifications, and the complications and clinical management of patients. We summarize also the main cellular and animal models developed to date and the possible future therapies.

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Bianchi, Paola, Anna Zaninoni, Elisa Fermo, Cristina Vercellati, Marcello Anna Paola, Alberto Zanella, Agostino Cortelezzi, and Wilma Barcellini. "Diagnostic Power of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) Evaluated in 118 Patients Affected By Hereditary Hemolytic Anemias." Blood 126, no. 23 (December 3, 2015): 942. http://dx.doi.org/10.1182/blood.v126.23.942.942.

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Abstract Introduction: Hereditary hemolytic anemias (HAs) is a group of heterogeneous disorders mainly due to defects of red cell (RBC) membrane and metabolism. HAs of RBC membrane are classified according to the morphological appearance on a blood smear: the more common are hereditary spherocytosis (HS), elliptocytosis, followed by stomatocytosis (dehydrated, DHSt and overhydrated, OHSt). Among RBC enzyme defects associated with chronic hemolysis, pyruvate kinase (PK) deficiency is the most common, followed by glucose-6-phosphate isomerase (GPI), pyrimidin 5' nucleotidase (P5'N), and more rarely, other enzymes deficiencies. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a battery of laboratory tests. Ektacytometry, and more recently, laser-assisted optical rotational cell analyser (LoRRca MaxSis, Mechatronics Instruments, NL), able to measure RBC deformability in osmotic gradient conditions (Osmoscan), is reported to be a useful tool in the detection of RBC membrane disorders and in particular for the differential diagnosis of HSt. Few data are available in other haemolytic anemias. Methods: We evaluated the diagnostic power of laser-assisted optical rotational cell analyser in 84 cases with RBC membrane disorders (69 HS, 7 HE, 7 DHSt, 1 OHSt) and 26 enzymopathies: 19 PK deficiency (14 not splenectomised), 4 GPI deficiency and 3 P5N deficiency. Moreover, we analysed 8 congenital diserythropoietic anemia type II (CDA II), of whom 3 not splenectomised. A reference curve interval was obtained from 89 healthy controls. The evaluated parameters were: Omin (osmotic value at which the EI reaches its minimum, coinciding with the 50% hemolysis in osmotic fragility assays), EImax (maximal deformability obtained near the isotonic osmolality), and Ohyper (osmotic value at which the EI reaches half of its maximum value, representing cellular hydration status). Results: Considering membrane defects, all the 69 HS, regardless the biochemical defect, showed typical altered ektacytometry curves, with a decreased EImax (0.516±0.05 vs 0.595±0.008 for controls, p<0.0001) and right shifted Omin (167±11.4 vs 143.7±7.2, p<0.0001); the 7 HE cases displayed a trapezoidal feature and a decreased EImax (0.547±0.05 vs 0.595±0.008, p<0.001). Among the hereditary stomatocytosis, the 7 DHSt were characterized by a left-shifted curve (Fig 1a). Concerning RBC enzymopathies, we found that all the GPI deficient cases showed altered enlarged Osmoscan curves associated with significant increased Ohyper (539±36 vs 462±13.4, p<0.0001), whereas not splenectomised patients with PK deficiency showed an Osmoscan curve characterised by an increased EImax (0.601±0.01 vs 0.595±0.008), although not significant (Fig 1b). Osmoscan curve in not splenectomised CDAII falls in the low reference area of normal controls. However, Omin and EImax were signifificantly different from HS patients thus permitting a differential diagnosis (Omin 149.3 ±3.0 vs 167±11.4, p<0.01; EImax 0.578±0.02 vs 0.516±0.05, p<0.05) (Fig 1c). We separately analysed splenectomised and not splenectomised patients. Interestingly, for some diseases (PK, P5N, CDAII and OHSt) Osmoscan curve after splenectomy, falls in a defined atypical area, regardless the pathology (Fig 1d). Conclusion: The Osmoscan curves were diagnostic for all the HS and HE cases analysed, with Omin and EImax parameters significantly different from controls. DHSt cases always showed typical left-shifted curve. Interestingly, Ohyper was firstly described increased in GPI deficiency, offering a new diagnostic tool for this rare enzyme defect. The interpretation of Osmoscan requires caution in splenectomised cases as splenectomy may interfere with the RBC deformability. In conclusion, the Osmoscan analysis performed by LoRRca Maxsis represents a useful and feasible first step screening test in the diagnosis of RBC membrane disorders and other rare haemolytic anemias. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Garufi, C., F. R. Spinelli, S. Mancuso, F. Ceccarelli, and F. Conti. "AB0704 TELEMEDICINE AT THE TIME OF COVID-19: THE EXPERIENCE WITH RA PATIENTS TREATED WITH JAK-INHIBITORS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 1384.1–1384. http://dx.doi.org/10.1136/annrheumdis-2021-eular.3892.

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Background:The spread of COVID-19, the lockdown, the limited access to care reevaluated the role of tele-consultation and self-assessment.Objectives:Our aim was to evaluate in a cohort of Rheumatoid Arthritis (RA) patients treated with JAK-inhibitors (JAKi): the self-assessed disease activity during lockdown, the lockdown impact on fatigue, anxiety, depression and the prevalence of Covid-19.Methods:We enrolled RA patients treated with baricitinib or tofacitinib. At baseline (BL) and follow-up we collected: patients’ demographic data, composite disease activity indices (CDAI, DAS28CRP), global assessment (PGA), pain visual analogue scale (VAS), FACIT (functional assessment of chronic illness therapy) and a self-rating scale for disease impact on anxiety and depression (Zung-A/D). Patients were instructed on how to perform self-assessment through video-material and fulfilled the online form of “Rheumatoid Arthritis Impact of Disease” (RAID)1 and “RA Disease Activity Index” (RADAI). To capture the pandemic effect, we compared patients in different status (remission, low, moderate and high-disease activity) at the last in-person visit (preCoV) through the DAS28CRP and CDAI, to the tele-health visit (THV), measured by the RAID. BL and pre-CoV ZUNG-A, ZUNG-D, FACIT questionnaires were compared with the online results during the pandemic. Exposure, tests and symptoms of Covid-19 were recorded. Data were expressed as mean±standard deviation or median(IQR) according to distribution.Results:Twenty patients (median age 58.2±11.9 and mean disease duration 153.5 ± 112.7 months) were treated with tofacitinib and 27 with baricitinib. The median time-lapse between the pre-CoV visit and the THV was 12 (IQR 4) weeks. DAS28CRP and CDAI significantly decreased from BL to pre-CoV visit. During the last in-person visit, 21 patients (48.83%) were in remission, 9 (20.93%) in low disease activity; according to the RAID, 15 (31.91%) and 7 (14.89%) patients were respectively in remission and low disease activity during the THV (Table A). PGA and pain significantly decreased from BL to pre-Cov visit but worsened during the lockdown (Table A). FACIT remaining stable during THV. At THV, we detected a significant improvement of anxiety from BL (Zung-A) and a tendency to lower depression scores compared to BL (Table A). JAKi showed a good safety profile considering Covid-19 symptoms, none of the patients was diagnosed with SarsCoV2 infection.Conclusion:This is the first study on virtual assessment in RA patients treated with JAKi. The unique social experiment of the pandemic impaired the clinical response already achieved before the lockdown, without a collateral worseling of FACIT, anxiety and depression.References:[1]Gossec L, et al. Ann Rheum Dis. 2009[2]Stucki G, et al. Arthritis Rheum. 1995Table A.DAS28, CDAI, RAID scores and patient-reported outcomes assessment at baseline and during the follow-upBLpre-CoVTHVDISEASE ACTIVITYN (%)N (%)N (%)REMISSIONDAS280 (0%)21 (48.8%)CDAI0 (0%)10 (22.7%)RAID15 (31.9%)LOW DISEASEDAS281(2.1%)9 (20.9%)CDAI7(14.8%)23 (52.2%)RAID7 (14.9%)MODERATEDAS2833 (70.2%)12 (27.9%)CDAI17 (37.1%)8 (18.1%)RAID13 (27.6%)HIGHDAS2813 (27.6%)1 (2.3%)CDAI23 (48.9%)3 (6.8%)RAID12 (25.5%)GH70 (30)20 (49.5)*45 (45)*#Pain70 (28)25 (45.5)*40 (48.5)*#Zung A37 (9)37 (10.2)35 (14)*Zung-D39 (17)39 (13)*38 (12)FACIT11.5 (17.2)8 (19.5)7(15)* p≤0.001 vs BL# p ≤0.04vs preCoVData expressed as median (IQR)Disclosure of Interests:Cristina Garufi: None declared, Francesca Romana Spinelli Speakers bureau: Abbvie, Eli Lilly, Consultant of: Gilead/Galapagos, Eli Lilly, Grant/research support from: Pfizer, Silvia Mancuso: None declared, Fulvia Ceccarelli: None declared, Fabrizio Conti Speakers bureau: Abbvie, Eli Lilly, Sanofi, Pfizer, Consultant of: Gilead/Galapagos

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Ramrattan, L. A., Z. Vaghaiwalla, S. Singh, K. Ramsubeik, M. Thway, and G. Kaeley. "AB0119 DISCORDANCE OF RAPID3 AND CDAI IN RHEUMATOID ARTHRITIS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 1088.2–1089. http://dx.doi.org/10.1136/annrheumdis-2021-eular.1682.

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Background:The Clinical Disease Activity Index (CDAI) and the Routine Assessment of Patient Index Data 3 (RAPID3) ascertain rheumatoid arthritis (RA) disease activity and inform treatment decisions. The CDAI has provider and patient components, whilst the RAPID3 only has patient driven measures. During the COVID-19 pandemic, telemedicine visits relied on RAPID3 as a clinical outcome measure and subsequently was incorporated into all clinical visits in addition to the CDAI. On an ad-hoc basis, discrepancies were noted for the disease activity level generated by these two measures. The purpose of this retrospective study was to formally analyze the relationship between these measures.Objectives:To determine the concordance of the outcome measures RAPID3 with CDAI in patients with established RA as a quality improvement project.Methods:This is a retrospective study of 49 patients that fulfilled the American College of Rheumatology 2010 criteria for Rheumatoid Arthritis. IRB approval was obtained. The medical records of patients seen between June to October 2020 at the rheumatology department at UF health were reviewed. Data collected included age, gender, race, number of years with RA, Rheumatoid factor (RF) and anti-citrullinated protein antibody (CCP Ab) positivity, disease modifying treatments, ESR and CRP as well as CDAI and RAPID3 scores as calculated by clinic staff. The charts were reviewed by the authors and RAPID3 scores were verified.Results:The population ranged from 35- 90 years and duration of RA from 1- 30 years. CCP Ab was present in 75% of patients and RF in 71%. Patients were on DMARDs either monontherapy (29%), dual therapy (60%) or triple therapy (10%). Antirheumatic medications used were plaquenil, methotrexate, leflunomide, etanercept, adalimumab, infliximab, tofacitinib, upadacitinib and rituximab. ESR range was 2-110 and CRP 0.2- 83.1. The CDAI and RAPID3 concordance was found to be 37% with RAPID3 being higher in 45% of patients. RAPID3 was lower only in 14% of patients. There was incorrect calculation of the RAPID3 26% of the time by clinic staff. Table 1 summarizes this data. Figure 1 shows RAPID3 and CDAI compared in scatterplots.Table 1.Patient PopulationAge35-90 yearsRaceAfrican American-13Caucasian-10Hispanic-3Not Hispanic=13RF Positive35/49 (71)%CCP Ab37/49 (75%)Both RF and CCP Ab Positive32/49 (65%)Patients on monotherapy14/48 (29%)Patients on dual therapy29/48 (60%)Patients on triple therapy5/48 (10)Antirheumatic Drugs usedPlaquenil, methotrexate, leflunomide, etanercept, adalimumab, infliximab, tofacitinib, upadacitinib, rituximabCDAI and RAPID3 Concordance18/49 (37%)RAPID 3 Higher than CDAI22/49 (45%)RAPID 3 lower than CDAI7/49 (14%)Incorrect Calculation of RAPID3 by clinic staff11/42 (26%)Figure 1.Scatterplots of three RAPID3 strata. Red dots represent discordant subjects when compared to CDAI. Note: Panel C demonstrates the subjects that were in low disease activity in red that had a high severity RAPID3 scoreConclusion:This study shows that RAPID3 may overestimate disease activity level for patients above low disease activity. Treatment escalation based on RAPID3 in discordant patients may be inappropriate. When making treatment decisions, a measure that includes objective physical examination and provider judgment is desirable.References:[1]Kumar, B. S., Suneetha, P., Mohan, A., Kumar, D. P. & Sarma, K. V. S. Comparison of Disease Activity Score in 28 joints with ESR (DAS28), Clinical Disease Activity Index (CDAI), Health Assessment Questionnaire Disability Index (HAQ-DI) & Routine Assessment of Patient Index Data with 3 measures (RAPID3) for assessing disease activity in patients with rheumatoid arthritis at initial presentation. Indian J Med Res146, S57–S62 (2017).[2]Pincus, T., Swearingen, C. J., Bergman, M. & Yazici, Y. RAPID3 (Routine Assessment of Patient Index Data 3), a Rheumatoid Arthritis Index Without Formal Joint Counts for Routine Care: Proposed Severity Categories Compared to Disease Activity Score and Clinical Disease Activity Index Categories. The Journal of Rheumatology35, 2136–2147 (2008).Disclosure of Interests:None declared

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Agrigento, Veronica, Sclafani Serena, Paolo Rigano, Rita Barone, Giuseppina Calvaruso, Rosario Di Maggio, Massimiliano Sacco, et al. "Congenital Dyserythropoietic Anemias: Molecular Diagnosis and Diagnostic Approach in a Cohort of Italian Patients." Blood 126, no. 23 (December 3, 2015): 2142. http://dx.doi.org/10.1182/blood.v126.23.2142.2142.

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Abstract Introduction. Congenital dyserythropoietic anemias (CDAs) are hereditary rare erythropoietic disorder characterized by distinct morphological abnormalities of the bone marrow cells, ineffective erythropoiesis and systemic iron overload. This conditions is characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. Three types of CDA are known: types 1, 2 and 3.The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division. We describe the clinical and laboratory manifestations, the diagnosis procedure, the therapeutic approaches and the clinical phenotype in some cases of congenital dyserythropoietic anemia (CDAs) in Italian patients. The molecular analysis allow us to identify several genetic variants some of which have never been described previously. This report highlights the importance of recognizing CDAI even in countries where thalassemia is common. Among the different tools genetic study of CDA-related genes remains the main approach for pursuing the right diagnosis. Methods. Peripheral blood samples from 100 normal adults and 20 patients with different types of anemia were collected. Blood samples were analyzed by EMA binding test and by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate ( SDS-PAGE). SDS PAGE was carried out in a 3.5-17% exponential gradient gel in Fairbanks buffer Genomic DNA was extracted from mononuclear cells of peripheral blood samples, by using a phenol-chloroform method. Polymerase chain reaction (PCR) products were sequenced directly using Big-Dye terminator 3.1 cycle sequencing kit and run on ABI PRISM 3130 DNA analyzer. The bone marrow of all patients was analysed with light or electron microscopy. The diagnosis of CDAs was based on the presence of mild to moderate anemia ineffective erythropoiesis and bone marrow erythroblasts morphological abnormalities. SEC23B, CDAN1, KLF1, BCL11A gene sequencing analysis was performed to highlight the presence of nucleotide variations and their relationship with the clinical presentation. Results and Discussion. We collected blood samples from 20 Italian patients with suspect of CDAs and 100 samples belonging to the healthy control subjects.None mutation in the genes analyzed was found in the samples controls.We identified SEC 23 B mutations in 4 out of 20 patients analysed. In two patients with suspect of CDAII we found two nucleotide variations; SDS PAGE in these patients identified the presence of abnormal band 3 and the EMA binding test resulted pathologic. Bone marrow (BM) light microscopy revealed more than 10% mature binucleated erythroblasts. In the patients suspected of CDA1, several gene variants were found in the CDAN1 gene, some described in the literature as mutations or polymorphisms, others of uncertain significance. In a patient diagnosed with CDA1 two novel mutations was found. EMA binding test resulted normal in all patients with CDA1. Light microscopy of the BM of CDA1 patients showed erythroid hyperplasia, presence of internuclear bridges between intermediate erythroblasts and characteristic pattern of spongy "swiss cheese" hetrochromatin in electron microscopy. None KLF1 and BCL11A mutations correlated with atypical CDAs was identified. In this last patients the EMA binding test resulted normal. CDAs are rare clinical hereditary disorders whose correct diagnosis is often delayed to adolescence or adulthood, when significant iron overload and end organ damage may have been occurred. Patients are often misdiagnosed with other congenital haemolytic anaemias. Inaccurate diagnosis can lead to inappropriate therapies, such as iron supplements, aggressive transfusion or splenectomy. However even if the gold standard for the CDAs diagnosis is the electronic microscopy, the identification of the mutated genes involved in the majority of CDAs patients made in recent years has improved the possibility of detecting these diseases. Disclosures No relevant conflicts of interest to declare.

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Yoshii, I. "AB0233 ATTAINING CDAI REMISSION IS THE FIRST GATEWAY TO ATTAIN BOOLEAN REMISSION." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1417.1–1417. http://dx.doi.org/10.1136/annrheumdis-2020-eular.1550.

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Background:Boolean remission is most stringent but most comparable remission status for the patient with rheumatoid arthritis (RA). Clinical remission evaluated with clinical disease activity index (CDAI) is also one of the most popular index for evaluation of RA treatment. These two criteria often overlap, but some are split.Objectives:Clinical significance of attaining CDAI remission before attaining Boolean remission was investigated.Methods:Patient with RA were treated in the institute since August 2010 under treat to target (T2T) strategy. In accordance with T2T, RA patients were monitored from the first consultation with parameter such as tenderness joint count (TJC), swollen joint count (SJC), patient’s global assessment (PGA), evaluator’s global assessment (EGA), C-reactive protein (CRP), modified Health Assessment Questionnaire (mHAQ), pain scale with visual analog scale (PS-VAS), and EuroQOL 5-dimension (EQ-5D). CDAI and Boolean are also evaluated at the same time. Radiographs of bilateral hands and feet are taken once a year from the first consultation, and the Sharp/van der Heijde Score (SHS) is measured.In patients, a group who attained CDAI remission prior to attaining Boolean remission (CDAI-R), a group who could not attain CDAI remission previously than attaining Boolean remission (CDAI-F), and a group who could not attain Boolean remission despite attaining CDAI remission (Boolean-F) were picked up and divided according to change of disease activity. Among these three groups, mean age, sex, education level, job style, anti-cyclic citrullinated polypeptide antibodies (ACPA), rheumatoid factor (RF), the CDAI score, the HAQ score, PS-VAS and quality of life index (QOL) calculated from EQ-5D were compared with each other using Mann-Whitney U-test. Boolean remission attaining rate whether CDAI remission attained was compared with chi square test.Results:Patient group configured with 255 of CDAI-R, 160 of CDAI-F, and 28 of Boolean-F. Patient who could not attain none of CDAI nor Boolean remission counted 175. In background factors at baseline, mean age, the HAQ score, and SHS of the Boolean-F were significantly older than the other groups. In the two groups of CDAI-R and CDAI-F, 28-joints disease activity score with C-reactive protein (DAS28-CRP), CDAI and PS-VAS in the CDAI-R were significantly lower than in the CDAI-F, similarly, DAS28-CRP, the CDAI score, the HAQ score, PS-VAS and QOL after Boolean remission attain were significantly higher in the CDAI-F than the CDAI-R. Sensitivity of Boolean remission when attaining CDAI remission previously before Boolean remission is 93.4%, and specificity was 52.2% (p<1.0x10-30).Conclusion:Attaining CDAI remission previously is extremely important, both for attaining Boolean remission and more stable clinical course after attaining Boolean remission. CDAI remission could be the first gateway to send sustainable QOL course.Disclosure of Interests:None declared

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Yoshii, I. "AB0232 PAIN SCORE WITH VISUAL ANALOG SCALE OF 30MM OR MORE IS A RISK FACTOR OF WORSENING CLINICAL DISEASE ACTIVITY INDEX (CDAI) AT THREE MONTHS AFTER ATTAINING CDAI REMISSION IN PATIENT WITH RHEUMATOID ARTHRITIS." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1416.1–1417. http://dx.doi.org/10.1136/annrheumdis-2020-eular.1916.

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Background:In treating with rheumatoid arthritis (RA), it is needless to say essential treatment goal with first priority. On the other hand, patient’s pain influences on clinical indices deeply, however, pain score is not been regarded as most important despite that correlates with patient reported outcome.Objectives:Clinical significance of remnant pain score for clinical outcome although attaining remission in clinical disease activity index (CDAI) statistically.Methods:RA patient who have attained remission with CDAI were picked up. These patients were divided into two groups whether CDAI at three month after the first CDAI remission attained; namely CDAI-R or CDAI-F. Background data such as sex, age at onset, age, anti-cyclic citrullinated polypeptide antibodies (ACPA), rheumatoid factor (RF), Sharp/van der Heijde Score (SHS), clinical disease activity score (CDAI), C-reactive protein (CRP), modified Health Assessment Questionnaire score (mHAQ), and pain score with visual analog scale (PS-VAS) at first consultation, time span from the first consultation to first CDAI remission were compared between the two groups using Mann-Whitney U-test. CDAI, CRP, mHAQ, PS-VAS, and QOL value calculated from EuroQOL-5 dimension questionnaire (EQ-5D) at the time of CDAI were also statistically compared with Mann-Whitney U-test. Parameters that demonstrated statistical significance within 5% were picked up, and odds ratio for CDAI remission were calculated with binary logistic regression analysis. Moreover, parameters that demonstrated statistical significance with p-value within 5% were evaluated with receiver’s observational characteristics (ROC) analysis, and cut-off index (COI) was calculated.Results:A total of 907 patients with 594 CDAI-R and 313 CAI-F were recruied. Demographic characteristics of the two groups were shown in Table 1. SHS at first consultation and time span from first consultation to CDAI remission attained demonstrated significantly less in the CDAI-R than the CDAI-F group, while the other parameters demonstrated no significant difference. CRP, CDAI, mHAQ, PS-VAS, and QOL at CDAI remission demonstrated significant difference between the CDAI-R and CDAI-F groups. With binary logistic regression analysis, CRP, CDAI, and PS-VAS demonstrated significant regression for CDAI-R with 1.68, 0.71, and 0.78 in odds ratio, respectively. COI for CDAI remission was 0.4, 1.0, and 30 for CRP (p=2.4 x 10-4), CDAI (p=3.0 x 10-32), and PS-VAS (p=2.4 x 10-4), respectively.Conclusion:PS-VAS at the moment of CDAI remission is suggested to be predictive factor for sustaining CDAI remission at three months thereafter as well as CRP value and the CDAI score.Table 1.Demographic characteristics of the two groupsCDAI-RCDAI-Fp-valuecases594313sex430 (72.4%)313 (80.2%)5.7 x 10-2age at onset62.260.91.8 x 10-1age at FC65.865.14.7 x 10-1ACPA at FC209.3 (83.9%)227.9 (86.7%)7.2 x 10-2RF at FC83.8 (92.2%)92.3 (86.1%)5.6 x 10-2SHS at FC41.966.96.0 x 19-5CDAI at FC10.711.15.5 x 10-2CRP at FC1.31.61.2 x 10-1mHAQ at FC0.4390.4753.2 x 10-1PS-VAS at FC32.634.92.1 x 10-1time span3.74.56.4 x 10-4Abbreviations: FC, first consultation; ACPA, anti-cyclic citrullinated polypeptide-antibodies; RF, rheumatoid factor; SHS, Sharp/van der eijde Score; CDAI, clinical disease activity index; CRP, C-reacive protein; mHAQ, modified Health Assessment Questionnaire; PS-VAS, pain score with visual analog scale; time span, time span from FC to date first CDAI remission attained.Disclosure of Interests:None declared

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Shafiq, Muhammad Amir, Muhammad Shafiq, and Nisar Ahmed. "Closed Loop Direct Adaptive Inverse Control for Linear Plants." Scientific World Journal 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/658497.

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In direct adaptive inverse control (DAIC), parameters of the controller are estimated directly in the feed-forward loop. In this paper, we propose a closed loop direct adaptive inverse control (CDAIC) scheme which improves tracking, error convergence, and disturbance rejection properties of DAIC. CDAIC is applicable to stable or stabilized, minimum or nonminimum phase linear plants. CDAIC and DAIC are compared using computer simulations for disturbance free and disturbed discrete type nonminimum phase linear plants. CDAIC shows better results compared to DAIC in terms of mean square tracking error and disturbance rejection.

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Singh, H., H. Kumar, R. Handa, P. Talapatra, S. Ray, and V. Gupta. "Use of Clinical Disease Activity Index Score for Assessment of Disease Activity in Rheumatoid Arthritis Patients: An Indian Experience." Arthritis 2011 (December 29, 2011): 1–5. http://dx.doi.org/10.1155/2011/146398.

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Introduction. Serial objective assessment of disease activity in Rheumatoid Arthritis (RA) is imperative to achieve remission. The CDAI score appears more practical than DAS-28 in routine assessment of disease activity in RA patients. Objective. To evaluate correlation and agreement of the DAS-28 with CDAI in RA patients. Methods. A total of 200 patients of RA were evaluated by DAS-28 and CDAI and divided into 4 categories of disease activity i.e. Group-I: Remission (DAS-28 < 2.6; CDAI < 2.8), Group II: Low disease activity (DAS-28 = 2.6–3.2; CDAI = 2.8–10), Group III: Moderate disease activity (DAS-28 = 3.2– 5.1; CDAI = 10–22), Group IV: High disease activity (DAS-28 > 5.1; CDAI > 22). DAS-28 was compared to CDAI in each group using spearman correlation coefficient and kappa statistics. Results. Group I shows mean DAS-28 of 1.99±0.38; mean CDAI of 0.90±0.65, (P = 0.0001). Group II shows mean DAS-28 of 3.04±0.17; mean CDAI of 6.45±02.35, (P = 0.0001). Group III shows mean DAS-28 of 4.25±0.58; mean CDAI of 16.46±3.31 (P < 0.0001). Group IV shows mean DAS-28 of 6.38±0.87; mean CDAI of 38.56±11.88 (P < 0.0001). Kappa statistics (κ) of the above comparison was 0.533. Conclusion. Our findings indicate that CDAI—a composite score that employs only clinical variables and omits assessment of Acute Phase Reactant (APR), has moderate to good correlation (Kappa value = 0.533) to DAS-28 for assessment of disease activity in RA patients.

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Moed, S. "Top Production and Properties at CDFII." Journal of Physics: Conference Series 110, no. 4 (May 1, 2008): 042016. http://dx.doi.org/10.1088/1742-6596/110/4/042016.

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Bianchi, Paola, Elisa Fermo, Jennifer C. Eng, Jacob C. Ulirsch, Cristina Vercellati, Paola Braidotti, Gordon Hildick-Smith, et al. "Biallelic Mutations in PARP4 Are Linked to a Variant Form of Congenital Dyserythropoietic Anemia." Blood 126, no. 23 (December 3, 2015): 272. http://dx.doi.org/10.1182/blood.v126.23.272.272.

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Abstract Congenital dyserythropoietic anemia (CDA) type II is the most frequent type of congenital dyserythropoietic anemia; it is transmitted in an autosomal recessive fashion and is characterized by ineffective erythropoiesis, peripheral hemolysis, bi-multinuclearity in the erythroblasts, and hypoglycosylation of red blood cell (RBC) membrane proteins such as band 3. The disease is generally caused by biallelic mutations in the SEC23B gene. However, there are a small portion of patients with clinical and hematologic features of CDA II that are negative for mutations in SEC23B, suggesting that alternative etiologies for such disturbed erythropoiesis exist. We identified two siblings of Italian origin who had dyserythropoiesis with a chronic macrocytic anemia. Their parents were healthy with normal hematologic parameters. No history of consanguinity for at least three generations was noted. The affected siblings had anisopoikylocytosis on peripheral blood smear with stomatocytes (8-9%), spherocytes (4-5%), rare ovalocytes, and dacryocytes. RBCs osmotic fragility was increased but the red cells had normal eosin-5-maleimide (EMA)-binding. Serum ferritin and transferrin saturation were increased in only one sibling. Bone marrow morphology revealed erythroid hyperplasia (myeloid: erythroid ratio = 0.6) with binuclearity and megaloblastic changes, as well as occasional cytoplasmic bridging between cells at different stage of maturation; electron microscopy of bone marrow erythroblasts showed multiple membranes that ran parallel to the plasma membrane or that were grouped in stacked segments, possibly attributable to residual endoplasmic reticulum (ER) cisternae. SDS-PAGE analysis of RBC ghosts from both siblings demonstrated hypoglycosylation of band 3 and GLUT1, as well as residual residual Protein Disulphide Isomerase (PDI) positive ER remnants, as observed in classical CDA II cases. However, in contrast to CDAII, the Ham's test performed with 15 normal serum samples was negative, and no mutations were detected in the SEC23B gene. To uncover the underlying etiologies, whole-exome sequencing was conducted on all available family members. After filtering for common variants, only a single gene had biallelic mutations in the affected siblings, which were transmitted from the unaffected heterozygous parents. The identified mutations resided in the PARP4 gene, which encodes a poly-ADP ribose polymerase enzyme, and were predicted to be deleterious. We demonstrate that knockdown of PARP4 using shRNA in primary human erythroid progenitors results in impaired erythroid differentiation and increased apoptosis. In addition, morpholino-mediated knockdown of the PARP4 orthologue in the zebrafish resulted in dyserythropoiesis and anemia in developing embryos. Sequencing of PARP4 in additional rare cases of CDA II without an identified molecular basis will help to uncover the frequency and spectrum of PARP4 mutations leading to dyserythropoiesis. The finding of a new gene implicated in a similar type of CDA with features such as redundant ER membranes offers the potential for more mechanistic dissection of the role of both SEC23B and PARP4 in erythroid development and suggests that new insight can be gained into the underlying pathophysiology of both normal and disordered erythropoiesis through the study of such rare cases. Disclosures No relevant conflicts of interest to declare.

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Galimand, Julie, Helene Bourdeau, Odile Fenneteau, Sophie Dreux, Nathalie Couque, Severine Drunat, Serge Pissard, et al. "Deciphering the Causal Diagnosis of Hydrops Fetalis or Unexplained Fetal Anemia Using Targeted Next Generation and Exome Sequencing." Blood 134, Supplement_1 (November 13, 2019): 3519. http://dx.doi.org/10.1182/blood-2019-127432.

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Fetal anemias are serious complications during pregnancies, which could lead to fetal death in case of hydrops fetalis (1/3000 pregnancies). Fetal anemia and hydrops fetalis are in most cases the result of fetal maternal alloimmunization, parvovirus B19 infection, fetal maternal hemorrhage, chromosomal abnormalities, congenital malformations, metabolic diseases, and in the context of hematological disorders, alpha-thalassemia. However, in one case out of 5, fetal anemia remains unexplained after an exhaustive first line of etiological evaluation. In order to identify the cause of the unexplained fetal anemia and to provide advice regarding prenatal diagnosis for next pregnancy, we have developed useful diagnostic tools on fetal blood based on erythrocyte and reticulocyte indices, examination of red cell morphology, flow cytometry (EMA test), osmotic gradient ektacytometry and molecular screening analysis. 43 fetal samples (30 probands) have been referred to our Hematology diagnostic lab in the time span between 2012-2018. In majority of the cases, various analyses were performed on fetal blood (23 out of 43) and in other instances during post-mortem examination following death (17 out of 43). Fetal blood purity was confirmed by microsatellite analysis on both parental and fetal DNAs. Informed consent was obtained from the mother in all cases. In 6 of 43 cases, prenatal diagnosis was performed after identification of the causal mutation responsible for the hydrops fetalis in the first fetus. Hydrops fetalis was suspected in 24 cases at the time of fetal sample collection. Red cell morphology and ektacytometry enabled the establishment of clinical diagnostic in two cases (congenital dyserythropoiesis type II (CDAII) and xerocytosis). Molecular screening analysis was performed by Sanger sequencing technique from 2012 to July 2016 and subsequently we designed a targeted Next Generation Sequencing (NGS) library including 74 genes involved in red cell disorders (n=9 fetuses) and exome sequencing (WES) was performed for 4 fetuses. Each identified allelic variation was confirmed by Sanger sequencing technique. Molecular Biology analysis (except the 6 prenatal diagnosis cases) was performed on 21 of 37 fetuses that enabled identification of the molecular defect in 10 fetuses. Rare red cell disorders were diagnosed in these cases including Diamond-Blackfan anemia (n=2), congenital dyserythropoiesis (n=6) and stomatocytosis (n=2) respectively. No putative pathogenous allelic variation following molecular screening could be identified in 4 fetuses. In the 6 cases which were screened for the molecular defect previously identified, none of the tested fetuses exhibited the allelic variation identified in the first fetus. In summary, targeted-NGS and WES are very valuable tools in the causal diagnosis of hydrops fetalis dues to unexplained anemia in addition to routine hematological tests (erythrocyte and reticulocyte indices, red cell morphology, flow cytometry, and ektacytometry) and after elimination of the most frequent causes of hydrops fetalis. This clinical problem is more frequent than has been previously surmised and needs more attention. Disclosures No relevant conflicts of interest to declare.

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Dan, J., J. Patel, G. Sprow, J. Concha, R. Feng, N. Kodali, T. Vazquez, D. Diaz, B. White, and V. Werth. "AB1485 PATIENT-REPORTED OUTCOMES AND BIOMARKERS ASSOCIATED WITH THE CUTANEOUS DERMATOMYOSITIS AREA AND SEVERITY ACTIVITY (CDASI-A) SCORE IN A PHASE 2 CLINICAL TRIAL IN DERMATOMYOSITIS." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 1847.1–1847. http://dx.doi.org/10.1136/annrheumdis-2022-eular.4986.

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BackgroundRetrospective reviews of clinical databases from two sites have identified strong relationships between patient-reported outcomes and skin activity in dermatomyositis (DM), as measured by CDASI-A.1,2 No studies validate these associations in a controlled setting. Additionally, the relationship between the PROMIS-29 Short Form and skin activity in DM has not been assessed. Previous investigations have demonstrated a correlation between IL-31 and itch in DM.3 IFN-β and IFN-γ are known type I and II interferons, which are critical drivers of DM pathogenesis.4ObjectivesTo assess correlations between CDASI-A, quality of life (QoL), and biomarkers of disease activity in a double-blind, randomized, placebo-controlled clinical trial.MethodsData were retrospectively collected from five visits of a Phase 2 trial evaluating Lenabasum, a cannabinoid receptor type 2 agonist. Quality of life assessments extracted from the trial included Patient Global Assessment (PtGA) scores, PROMIS domains, and Skindex domains. Skindex question 10, regarding itch, was included in the analysis as a separate domain. Physician Global Assessment scores were also evaluated. Additionally, biomarkers derived from skin samples via IHC/PCR collected at visits 1 and 6 were assessed for predictors of CDASI-A response and association with disease activity. Analysis used linear mixed effect models to account for within subject-variability and repeated measures, where applicable. Analysis was performed without regard to treatment arm, as our goal was to correlate CDASI, QoL, and biomarkers among all subjects.ResultsData from 22 subjects with DM and a combined total of 110 visits were included. Biopsies were collected from 12 subjects. Improvement in CDASI-A significantly correlated with Skindex-S, Skindex-E, Skindex-F, Skindex-Itch, PtGA global skin, PtGA global skin, PtGA global skin, and PtGA global skin, with p < 0.001. Improvement in PROMIS social role (p = 0.046) correlated with improvement in CDASI-A. Worsening of PROMIS fatigue (p = 0.019) and pain (p < 0.001) correlated with improvement in CDASI-A. Decreases in PGA overall disease, PGA skin activity, and PGA global skin all correlated with improvement of CDASI-A (p < 0.001). Change in IL-31 protein area positively correlated with change in disease activity (p = 0.047). A positive relationship between changes in IFN-β and IFN-γ protein area and disease activity trended towards significance.ConclusionIn accordance with previous investigations from our group, well-established measures of QoL correlated significantly with CDASI-A. These findings support that CDASI-A reflects both clinical and patient-reported aspects of skin disease and is an appropriate outcome in DM clinical trials. Additionally, Skindex and PtGA scores may better relate to skin activity as measured by the CDASI compared to PROMIS domains. IL-31, a cytokine previously associated with itch in DM,3 correlated significantly with CDASI-A in our study. Trends for IFN-β and IFN-γ reduction with disease improvement support their role in the pathogenesis of DM. This study helps define patient-reported outcomes and biomarkers that may be informative in DM trials.References[1]Goreshi R, et al. J Am Acad Dermatol. 2011;65(6):1107-1116[2]Robinson ES, et al. Br J Dermatol. 2015;172(1):169-174.[3]Patel J, et al. J Invest Dermatol. 2021;141(9):2151-2160.[4]Wong D, et al. PLoS One. 2012;7(1):e29161Disclosure of InterestsJoshua Dan: None declared, Jay Patel: None declared, Grant Sprow: None declared, Josef Concha: None declared, Rui Feng: None declared, Nilesh Kodali: None declared, Thomas Vazquez: None declared, DeAnna Diaz: None declared, Barbara White Shareholder of: Corbus Pharmaceuticals, Victoria Werth Speakers bureau: University of Pennsylvania, which owns the copyright for the CDASI, Grant/research support from: Corbus Pharmaceuticals

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Keystone, E., M. Movahedi, A. Cesta, C. Bombardier, J. Sampalis, and E. Rampakakis. "AB0211 DIFFERENTIAL INFLUENCE OF CDAI COMPONENTS BASED ON DISEASE STATE IN RHEUMATOID ARTHRITIS PATIENTS: REAL-WORLD RESULTS FROM THE ONTARIO BEST PRACTICES RESEARCH INITIATIVE (OBRI)." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1406.2–1406. http://dx.doi.org/10.1136/annrheumdis-2020-eular.3913.

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Background:Treat-to-target recommendations for rheumatoid arthritis (RA) dictate that remission or low disease activity should be aimed. Although numerous composite indices are available, the clinical disease activity index (CDAI) is commonly used in routine clinical care due to its simplicity and non-reliance on acute phase reactants.Objectives:The purpose of this analysis was to evaluate the CDAI properties both cross-sectionally and longitudinally in a cohort of RA patients followed in Canadian routine care.Methods:RA patients enrolled in the Ontario Best Practices Research Initiative (OBRI), with available follow-up for ≥6 months and data on CDAI, disease activity score based on 28 joints (DAS28), health assessment questionnaire (HAQ), and ACR/EULAR Boolean remission were included. For both the CDAI score and its change from baseline to 6 months, construct validity was assessed with principal component analysis, internal consistency with the Cronbach’s alpha coefficient (α), correlational validity with the Spearman’s rho coefficient, agreement in disease state classification with percent concordant pairs and the kappa statistic. Stratified analysis by presence of CDAI low disease activity (LDA) or remission was performed.Results:1,582 patients met the inclusion criteria. Principal component analysis showed that CDAI could be reduced to a single component when CDAI is >10 with SJC28 accounting for most variance in score and patient global assessment (PtGA) the least; whereas, when CDAI is ≤10, two distinct components were identified, the first comprising PtGA and physician global assessment (PhGA) and the second SJC28 and TJC28. In terms of internal consistency, high levels were observed for both CDAI at baseline (α=0.83) and its change from baseline to 6 months (α=0.81); however, the consistency between CDAI components was very low when CDAI is ≤10 (α=0.23).Overall, a strong positive correlation was observed between CDAI and DAS28 (rho=0.86) and their changes (rho=0.87) while its correlation with HAQ was weak. When stratifying by CDAI levels, the correlation of CDAI with DAS28 was moderate when CDAI is ≤10 and very weak when CDAI is ≤2.8. Similarly, agreement in the classification of LDA between CDAI and DAS28 or HAQ was fair to moderate, and agreement in classification of remission was poor to fair.Conclusion:CDAI and DAS28 correlate well when disease activity is moderate or high and poorly in LDA or remission. PtGA had a stronger influence on CDAI at LDA or remission state compared to moderate or high disease state. Thus, careful interpretation of PtGA is necessary particularly in patients who are identified as CDAI non-remitters.Disclosure of Interests:Edward Keystone Grant/research support from: AbbVie, Amgen, Bristol-Myers Squibb, F. Hoffmann-La Roche Inc, Gilead, Janssen Inc, Lilly Pharmaceuticals, Pfizer Pharmaceuticals, Sanofi-Aventis, Consultant of: AbbVie, Amgen, AstraZeneca Pharma, Biotest, Bristol-Myers Squibb Company, Celltrion,Crescendo Bioscience, F. Hoffmann-La Roche Inc, Genentech Inc, Gilead, Janssen Inc, LillyPharmaceuticals, Merck, Pfizer Pharmaceuticals, Sandoz, UCB., Speakers bureau: Amgen, AbbVie, Bristol-Myers Squibb Canada, F. Hoffmann-La Roche Inc., Janssen Inc., Merck, Pfizer Pharmaceuticals, Sanofi Genzyme, UCB, Mohammad Movahedi Consultant of: Allergan, Angela Cesta: None declared, Claire Bombardier Grant/research support from: Dr Bombardier reports sources of funding for Ontario Best Practice Research Initiative Research grants from Abbvie, Janssen, Amgen, Medexus, Merck, Pfizer, and Novartis outside of the submitted work. Consulting Agreements: Abbvie, Covance, Janssen, Merck, Pfizer, Sanofi and Novartis outside of the submitted work. Advisory Board Membership: Hospira, Sandoz, Merck, Pfizer and Novartis outside of the submitted work., John Sampalis: None declared, Emmanouil Rampakakis: None declared

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Yamaoka, K., S. B. Cohen, N. Sugiyama, H. Shi, J. L. Rivas, A. Diehl, and J. S. Smolen. "POS0650 PREDICTORS OF DURABLE CLINICAL RESPONSE TO TOFACITINIB 11 MG ONCE DAILY WITH OR WITHOUT METHOTREXATE IN PATIENTS WITH RHEUMATOID ARTHRITIS: POST HOC ANALYSIS OF DATA FROM A PHASE 3b/4 METHOTREXATE WITHDRAWAL STUDY." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 563.2–564. http://dx.doi.org/10.1136/annrheumdis-2021-eular.357.

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Background:ORAL Shift, a global Phase 3b/4 non-inferiority study, demonstrated sustained efficacy and safety of tofacitinib modified-release (MR) 11 mg once daily (QD) following methotrexate (MTX) withdrawal in patients with rheumatoid arthritis (RA) who achieved Clinical Disease Activity Index (CDAI) low disease activity (LDA) after treatment with tofacitinib + MTX.1Objectives:To assess predictors of durable clinical response in patients receiving tofacitinib MR 11 mg QD in ORAL Shift.Methods:ORAL Shift (NCT02831855) enrolled patients aged ≥18 years with moderate to severe RA and an inadequate response to MTX. Patients received open-label tofacitinib MR 11 mg QD + MTX for 24 weeks. Patients achieving LDA (CDAI score ≤10) at Week (W)24 entered the 24-week double-blind MTX withdrawal phase and were randomised 1:1 to receive tofacitinib MR 11 mg QD + placebo (tofacitinib monotherapy; ie blinded MTX withdrawal) or continue tofacitinib + MTX. In this post hoc analysis of randomised patients, we assessed predictors of durable response (maintenance of response from W24–48) per CDAI LDA and remission (CDAI score ≤2.8) criteria. All covariates were initially assessed for significance in a univariate logistic regression. Highly correlated covariates were reviewed to assess which would be removed prior to modelling in a multivariable logistic regression. Remaining significant (p≤0.10) covariates in the univariate regression were selected in the model using a stepwise selection process with p≤0.15 entry and p≤0.05 stay criteria. From the final model, estimated odds ratios (ORs) with 95% confidence intervals (CIs) are presented.Results:In the double-blind phase of ORAL Shift, durable CDAI LDA and remission rates were: 66.2% and 14.7%, respectively, with tofacitinib + MTX (N=266); and 55.3% and 11.0%, respectively, with tofacitinib + placebo (N=264) (Table 1). In the multivariable analysis, five patient covariates significantly predicted durable CDAI LDA (Figure 1; discussed hereafter). Each unit increase in CDAI score at W24 reduced the likelihood of maintaining CDAI LDA by 22.0%. Each unit increase in C-reactive protein (CRP) at W24 increased the likelihood of maintaining CDAI LDA by 4.0%; this may have been due to imbalanced CRP levels at W24 (randomisation) between treatment groups (Figure 1, footnote c). The odds of durable CDAI LDA were 53.0% lower in the US vs Europe and 61.0% lower in the US vs ‘other’ regions. Each unit increase in baseline Health Assessment Questionnaire-Disability Index (HAQ-DI) score reduced the odds of durable CDAI LDA by 34.0%. Patients receiving tofacitinib + MTX had 66.0% greater odds of durable CDAI LDA vs patients receiving tofacitinib + placebo. CDAI at W24 was the only significant predictor of durable CDAI remission in the multivariable analysis: OR (95% CI) 0.32 (0.24, 0.43); p<0.0001. Each unit increase in CDAI score at W24 reduced the odds of durable CDAI remission by 68.0%.Table 1.Durable CDAI LDA and remissiona in patients receiving tofacitinib MR 11 mg QD with MTX or placebo in the double-blind phase of ORAL ShiftTofacitinib + MTX(N=266)Tofacitinib + placebo(N=264)Durable CDAI LDA, n (%)176 (66.2)146 (55.3)Durable CDAI remission, n (%)39 (14.7)29 (11.0)aDurable CDAI LDA or remission was defined as achievement of LDA (CDAI score ≤10) or remission (CDAI score ≤2.8), respectively, at W24–48N, number of patients in each group; n, number of patients achieving outcomeConclusion:This post hoc analysis of data from ORAL Shift found that CDAI and CRP at W24, geographic region, baseline HAQ-DI and treatment could be predictors for durable CDAI LDA. As these findings were limited to patients who achieved CDAI LDA at W24 with tofacitinib MR 11 mg QD + MTX, additional data in the general patient population need to be investigated.References:[1]Cohen et al. Lancet Rheumatol 2019; 1: E23-34.Acknowledgements:Study sponsored by Pfizer Inc. Medical writing support was provided by Sarah Piggott, CMC Connect, and funded by Pfizer Inc.Disclosure of Interests:Kunihiro Yamaoka Speakers bureau: Actelion, Astellas, Chugai, Eisai, Eli Lilly, GlaxoSmithKline, Janssen, Mitsubishi Tanabe, Nippon Shinyaku, Pfizer Inc, Takeda, Consultant of: Actelion, Astellas, Chugai, Eisai, Eli Lilly, GlaxoSmithKline, Janssen, Mitsubishi Tanabe, Nippon Shinyaku, Pfizer Inc, Takeda, Stanley B. Cohen Consultant of: AbbVie, Eli Lilly, Genentech, Gilead Sciences, Pfizer Inc, Grant/research support from: AbbVie, Eli Lilly, Genentech, Gilead Sciences, Pfizer Inc, Naonobu Sugiyama Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Harry Shi Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Jose Luis Rivas Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Annette Diehl Shareholder of: Pfizer Inc, Employee of: Pfizer Inc, Josef S. Smolen Consultant of: AbbVie, AstraZeneca, Celgene, Celltrion, Chugai, Eli Lilly, Gilead Sciences, ILTOO, Janssen, Novartis-Sandoz, Pfizer Inc, Roche, Samsung, Sanofi, Grant/research support from: AbbVie and AstraZeneca

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Harrold, Leslie R., George W. Reed, Ashwini Shewade, Robert Magner, Katherine C. Saunders, Ani John, Joel M. Kremer, and Jeffrey D. Greenberg. "Effectiveness of Rituximab for the Treatment of Rheumatoid Arthritis in Patients with Prior Exposure to Anti-TNF: Results from the CORRONA Registry." Journal of Rheumatology 42, no. 7 (May 1, 2015): 1090–98. http://dx.doi.org/10.3899/jrheum.141043.

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Objective.To characterize the real-world effectiveness of rituximab (RTX) in patients with rheumatoid arthritis.Methods.Clinical effectiveness at 12 months was assessed in patients who were prescribed RTX based on the Clinical Disease Activity Index (CDAI). Change in CDAI was calculated (CDAI at 12 mos minus at initiation). Achievement of remission or low disease activity (LDA; CDAI ≤ 10) among those with moderate/high disease activity at the time of RTX initiation was compared based on prior anti-tumor necrosis factor agent (anti-TNF) use (1 vs ≥ 2) using logistic regression models.Results.Patients (n = 265) were followed for 12 months with a mean change in CDAI of −8.1 (95% CI −9.8 – −6.4). Of the 218 patients with moderate/high disease activity at baseline, patients with 1 prior anti-TNF (baseline CDAI 25.0) demonstrated a mean change in CDAI of −10.1 (95% CI −13.2 – −7.0); patients with ≥ 2 prior anti-TNF (baseline CDAI 30.0) demonstrated a mean change of −10.5 (95% CI −12.9 – −8.0). The unadjusted OR for achieving LDA/remission in patients with moderate/high disease activity at baseline exposed to ≥ 2 versus 1 prior anti-TNF was 0.40 (95% CI 0.22–0.73), which was robust to 4 different adjusted models (OR range 0.38–0.44).Conclusion.A good clinical response was observed in all patients; however, patients previously treated with 1 anti-TNF, who had lower baseline CDAI and a greater opportunity for clinical improvement compared with patients previously treated with ≥ 2 anti-TNF, were more likely to achieve LDA/remission.

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Hu, Fu, Qian Li, Benwei Zhu, Fang Ni, Yun Sun, and Zhong Yao. "Effects of module truncation on biochemical characteristics and products distribution of a new alginate lyase with two catalytic modules." Glycobiology 29, no. 12 (May 18, 2019): 876–84. http://dx.doi.org/10.1093/glycob/cwz064.

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Abstract In this work, we investigated the functions of structural modules within alginate lyase by truncating an endo-type alginate lyase into two successive catalytic modules. The effects of module deletion on biochemical characteristics and product distributions were further investigated. The N-terminal module (Aly7B-CDI) exhibited no activity toward alginate, polyM or polyG, but the C-terminal module (Aly7B-CDII) retained its activity. The full-length enzyme (Aly7B) and its truncated counterpart (Aly7B-CDII) had similar substrate specificities, but Aly7B-CDII had lower activity. Moreover, the activity of Aly7B was much higher than Aly7B-CDII at 30°C. Aly7B-CDII, however, possessed higher optimal pH and better pH stability than the full-length enzyme. The final degradation products for Aly7B were unsaturated di-, tri- and tetra-oligosaccharides, and those for Aly7B-CDII were unsaturated mono-, di-, tri-, tetra- and penta-oligosaccharides. Therefore, the potential impact of the noncatalytic module Aly7B-CDI on the catalytic module Aly7B-CDII was further elucidated by characterizing Aly7B and its truncations. These data contribute to the functional understanding of these differing modules.

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Wallbank, Andrew I., Terry P. Lebold, Aneta Borecki, Alexander M. Polgar, Blake M. Waters, Mark S. Workentin, and John F. Corrigan. "ZnII and CdII Ferrocenechalcogenolate Complexes." European Journal of Inorganic Chemistry 2017, no. 2 (September 21, 2016): 372–77. http://dx.doi.org/10.1002/ejic.201600898.

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Wang, Guang Hui, Da Jun Song, Hong Xiao Tian, Jian Gao, and Yu Liu. "Structure Studies on Order Assemblage of Cadmium^II Isophthalicacid Benzimidazole Complexes." Applied Mechanics and Materials 713-715 (January 2015): 2872–75. http://dx.doi.org/10.4028/www.scientific.net/amm.713-715.2872.

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The CadmiumII Isophthalicacid Benzimidazole complex [Cd2(C8H4O4)2 (C7H6N2)4(H2O)3], Triqua-bis (isophthalicacidato-O,O')-tetra (benzimidazole) bis (CadmiumII),the CdII atom is coordinated by two isophthalicacid anions and two benzimidazole and one water molecule in a distorted octahedral configuration with six-coordinations geometry. And one isophthalicacid ligand chelates to the CdII atom through its one carboxylic O atoms, but the other isophthalicacid ligand chelates to the CdII atom through its two carboxylic O atoms. The same as another CdII atom. So we get a Binuclear CdII metal complexe. The fact clearly suggests not so much significant contribution from the electrostatic interaction in the Cd-O bonding in bidentate Binuclear CdII metal complexes as we gotten in mononuclear MnII metal complexes. Adjacent complex link to each other via hydrogen bonds forming the three-dimensional supramolecular structure.

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Cané, Mariana. "La construcción discursiva de la inevitabilidad en los inicios del gobierno de la Alianza (Argentina, 1999-2000)." Papel Político 23, no. 2 (April 8, 2019): 1–23. http://dx.doi.org/10.11144/javeriana.papo23-2.cdii.

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Desde los inicios del gobierno de la Alianza (Diciembre 1999), gran parte de los discursos políticos establecieron polémicas en torno al significante “crisis”. En ese contexto, fueron enunciados una variedad de diagnósticos que disputaron por definir qué era lo que estaba en crisis, cuáles eran las causas de la misma y cómo debía ser conjurada. A partir del modo en que los discursos políticos delinearon los contornos de “la crisis”, hemos identificado tres grandes tópicas o conjuntos de argumentos (que denominamos fiscalista, asistencialista y mercadointernista) y un eje en disputa clave vinculado a la construcción de la inevitabilidad de las medidas a adoptar. Aunque estas tópicas signaron el campo discursivo de lo político hasta fines del año 2001, se consolidaron con sus principales rasgos en el marco de la polémica por la reducción salarial y la aprobación de la reforma laboral de mediados del año 2000. En este sentido, sostenemos que es clave rastrear la construcción discursiva de la inevitabilidad porque constituye un elemento central para comprender parte del proceso de pérdida de legitimidad de la palabra política que alcanzó su punto de mayor algidez en lo que se conoció como “la crisis del 2001”.

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He, Huiqin, Xin Chen, Da Miao, Hongxia Zhang, Yu Wang, Xingkang He, Xiaoli Chen, and Ning Dai. "Composite Dietary Antioxidant Index and Plasma Levels of Soluble Klotho: Insights from NHANES." Oxidative Medicine and Cellular Longevity 2023 (February 7, 2023): 1–8. http://dx.doi.org/10.1155/2023/3524611.

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Objectives. The association between dietary antioxidants and soluble Klotho (S-Klotho) levels remains unknown. We investigated to explore whether the composite dietary antioxidant index (CDAI) was associated with serum levels of S-Klotho in the middle-aged population. Methods. Eligible participants were identified from the National Health and Nutrition Examination Surveys (NHANES) from 2007 until 2016. The CDAI was calculated from the intake of six dietary antioxidants. The serum levels of S-Klotho were measured via enzyme-linked immunosorbent assay (ELISA). Generalized linear and nonlinear models were established to analyze the relationship between CDAI and S-Klotho levels. Results. Based on the S-Klotho quartiles, S-Klotho levels were higher in young women, Blacks, higher education, never smokers, lower waistlines, no medication use, and those with higher CDAI. Univariate analysis revealed that age, gender, race, smoking status, body mass index, waistline, and medication use were associated with serum levels of S-Klotho. When potential confounders were controlled, CDAI was significantly associated with S-Klotho levels. Subgroup analysis also revealed that this association remained significant in individuals who had the highest quartiles of CDAI, aged population (>60 years), male, and never smoker. A nonlinear relationship was observed between the CDAI and S-Klotho plasma concentrations. Conclusion. CDAI was positively correlated with plasma levels of S-Klotho after controlling for covariates. Further studies are needed to validate the current findings and explore the fundamental mechanisms.

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Dissanayake, Keerthie, Chandrika Jayasinghe, Priyani Wanigasekara, Jayampathy Dissanayake, and Ajith Sominanda. "Validity of clinical disease activity index (CDAI) to evaluate the disease activity of rheumatoid arthritis patients in Sri Lanka: A prospective follow up study based on newly diagnosed patients." PLOS ONE 17, no. 11 (November 29, 2022): e0278285. http://dx.doi.org/10.1371/journal.pone.0278285.

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Routine use of the Disease Activity Score-28 (DAS28) to assess the disease activity in rheumatoid arthritis (RA) is limited due to its dependency on laboratory investigations and the complex calculations involved. In contrast, the clinical disease activity index (CDAI) is simple to calculate, which makes the "treat to target" strategy for the management of RA more practical. We aimed to assess the validity of CDAI compared to DAS28 in RA patients in Sri Lanka. A total of 103 newly diagnosed RA patients were recruited, and their disease activity was calculated using DAS 28 and CDAI during the first visit to the clinic (0 months) and re-assessed at 4 and 9 months of follow-up visits. The validity of the CDAI, compared to DAS 28, was evaluated. Patients had a female preponderance (6:1) and a short symptom duration (mean = 6.33 months). Internal consistency reliability of CDAI, as assessed by Cronbach’s α test, was 0.868. Convergent validity was assessed by correlation and Kappa statistics. Strong positive correlations were observed between CDAI and DAS 28 at the baseline (0 months), 4 and 9 months of evaluation (Spearman’s r = 0.935, 0.935, 0.910, respectively). Moderate-good inter-rater agreements between the DAS-28 and CDAI were observed (Weighted kappa of 0.660, 0.519, and 0.741 at 0, 4, and 9 months respectively). Discriminant validity, as assessed by ROC curves at 0, 4th, and 9th months of the evaluation, showed the area under the curve (AUC) of 0.958, 0.979, and 0.910, respectively. The suggested cut-off points for different CDAI disease activity categories according to ROC curves were ≤ 4 (Remission), > 4 to ≤ 6 (low), > 6 to ≤ 18 (moderate), > 18 (high). These findings indicate that the CDAI has good concordance with DAS 28 in assessing the disease activity in RA patients, in this study sample.

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Ke, Xi-Jun, Dong-Sheng Li, Jun Zhao, Cai-Xia Meng, Xiao-Ning Zhang, Qiu-Fen He, Cai Li, and Yao-Yu Wang. "In situ attained CdII-coordination polymer with (3,4,9)-connected topology based on trinuclear CdII clusters." Inorganic Chemistry Communications 13, no. 4 (April 2010): 484–87. http://dx.doi.org/10.1016/j.inoche.2010.01.014.

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Journal articles on the topic 'CDAII'

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