Serum copper decrease and cerebellar atrophy in patients with nitrous oxide-induced subacute combined degeneration: two cases report

Background Subacute combined degeneration (SCD) is a neurological complication commonly associated with vitamin B12 deficiency. It can result from nitrous oxide (N2O) abuse and cause neuropsychiatric symptoms. However, there has been no literature regarding alterations of serum copper and cerebellum in SCD patients. Case presentation We reported two cases of young SCD patients with histories of N2O abuse. In these cases, elevated homocysteine, macrocytic anemia, spinal cord abnormalities, and peripheral nerve injuries were detected. In addition, decreased serum copper level and cerebellar atrophy were reported for the first time. The patients’ symptoms improved after withdrawal of N2O exposure and vitamin B12 supplements. Conclusion We reported two SCD cases with serum copper alteration and cerebellar atrophy after N2O abuse for the first time. These might be crucial complements to the diagnosis of SCD.

Autosomal Recessive Congenital Dyserythropoietic Anemia Type III Is Caused By Mutations in the Centralspindlin RACGAP1 Component

An autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III) is caused by a missense mutation in the KIF23 gene whose protein product, mitotic kinesin-like protein (MKLP1), is part of the centralspindlin complex involved in cytokinesis. Several case reports suggested the existence of an autosomal recessive inheritance form of CDA III so far not genetically characterized. By means of whole exome sequencing in a Spanish CDA III family with healthy parents, we identified in the male proband a novel homozygous missense mutation p.Pro432Ser in the RACGAP1 gene, which encodes for the RACGAP1 protein (Rac GTPase-activating protein 1, also known as MgcRacGAP or CYK-4), the partner of MKLP1 in the centralspindlin complex. A second CDA III Spanish patient has a different rare and novel homozygous missense mutation, p.Thr220Ala, in the RACGAP1 gene. Both patients presented with macrocytic anemia, aberrant multinucleated erythroblasts in the bone marrow typically seen in CDA III cases, no iron overload and skull defects secondary to severe anemia. Silencing of RACGAP1 using siRNA in HeLa cells mimics the cytokinesis defect observed in the bone marrow of our patients. Both mutations disrupt normal cytokinesis and alter the GTPase balance in patients' cells. We conclude that the autosomal recessive form of CDA type III is caused by mutations in the RACGAP1 gene, encoding for RACGAP1 protein, which is the partner of MKLP1 in the centralspindlin complex critical for cytokinesis and now both proteins are associated with CDA type III. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Interleukin-1/Toll-like Receptor Inhibition Can Restore the Disrupted Bone Marrow Microenvironment in Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice. Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors. Results: Transplant of NHD13 BM cells into aged IL1R1 wt recipients (NHD13→IL1R1 wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ko recipients (NHD13→IL1R1 ko). IL1R1 wt developed macrocytic anemia compared to IL1R1 ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p<0.05). In CFU-C assays, NHD13→IL1R1 wt and NHD13→IL1R1 ko demonstrated similar levels of CFU-activity whereas CFU-fibroblast (CFU-F) assays of IL1R1 wt recipients demonstrated lower numbers of large colonies (reported to contain highly proliferative mesenchymal stem cells (MSC)). Flow cytometry analysis of hematopoietic stem and progenitor cells (HSPC) population in IL1R1 wt recipients showed increased myeloid progenitors and decreased long-term HSC (LT-HSC) compared to IL1R1 ko recipients. Cytokine/chemokine profiles revealed that the inflammatory cytokines (IL-6 and TNFα) and macrophage activating cytokines (MCP-1 and M-CSF) were significantly decreased in NHD13→IL1R1 ko compared to NHD13→IL1R1 wt. Next, we treated MDS model mice with inhibitors. The pharmacological targeting of IL1R1 signaling in vivo was associated with decreased NHD13 cell burden and improvement of macrocytic anemia. CFU-C assays demonstrated some decreases in NHD13 CFU capacity post-treatment. Interestingly, non-NHD13 HSPCs assessed by CFU capacity increased in IRAK4 inhibitor treated mice. Consistent with this, flow cytometric analyses of HSPC pools demonstrated decreased NHD13 HSPCs (LT-HSC and granulocyte-monocyte progenitor cells) and increased non-NHD13 HSCs and myeloid progenitors compared to vehicle group. BMME cell populations showed that arteriolar endothelial cells and MSCs were also affected by drug treatment and both IL1R antagonist and MCC950 increased the number of large CFU-F colonies. Analysis of BMEF revealed the decreased IFNγ and IL-18 and upregulated M-CSF in MCC950 treated group. The cytokines of human BM serum revealed higher concentrations of soluble formed IL1R1, IL-18, CXCL1, and osteopontin in MDS compared to young or aged normal donors. Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME. Disclosures No relevant conflicts of interest to declare.

A clinical, histopathological, and molecular study of two cases of VEXAS syndrome without a definitive myeloid neoplasm

VEXAS (vacuoles, E1 enzyme, X- linked, autoinflammatory, somatic) syndrome is caused by somatic mutations in UBA1 and is identified using a genotype-driven method. This condition connects unrelated men with adult-onset inflammatory syndromes in association with hematologic manifestations of peripheral cytopenia and bone marrow myeloid dysplasia. While bone marrow vacuolization restricted to myeloid and erythroid precursors has been identified in VEXAS patients, the detailed clinical and histopathological features of peripheral blood and bone marrows remain unclear. The current case report describes the characteristic hematologic findings in patients with VEXAS, including macrocytic anemia, thrombocytopenia, marked hypercellular marrow with granulocytic hyperplasia, megaloblastic changes in erythroid precursors, and the absence of hematogones in addition to prominent vacuoles in myeloid and erythroid precursor cells. Characterizing the clinical and hematologic features helps to raise awareness and improve diagnosis of this novel, rare, but potentially under-recognized disease. Prompt diagnosis expands the general knowledgeable and understanding of this disease, and optimal management might prevent patients from developing complications related to this refractory inflammatory syndrome and improve the overall clinical outcome.

Nonlinear Relationship Between Macrocytic Anemia and Decompensated Hepatitis B Virus Associated Cirrhosis: A Population-Based Cross-Sectional Study

Background: Mean corpuscular volume (MCV) is major used as an indicator for the differential diagnosis of anemia. Macrocytic anemia in decompensated cirrhosis is common. However, the relationship between macrocytic anemia and decompensated hepatitis B virus (HBV) associated cirrhosis has not been fully addressed.Methods: In this cross-sectional study, a total of 457 patients diagnosed decompensated HBV associated cirrhosis who met all inclusion criteria from 2011 to 2018 were analyzed. Association between macrocytic anemia and the liver damaged (Model for End Stage Liver Disease (MELD) score) were examined using multiple logistic regression analyses and identified using smooth curve fitting.Results: Compared with normocytic anemia, MCV and MELD are significantly positively correlated in macrocytic anemia (p < 0.001). A non-linear relationship of MCV and MELD association was found though the piecewise linear spline models in patients with decompensated HBV associated cirrhosis. MCV positive correlated with MELD when the MCV was greater than 98.2 fl (regression coefficient = 0.008, 95% CI 0.1, 0.4).Conclusion: Macrocytic anemia may be a reliable predictor for mortality because it is closely related to the degree of liver damage in patients with decompensated HBV associated cirrhosis.

CLINICOETIOLOGICAL EVALUATION OF THE PATIENTS OF MACROCYTIC ANEMIA PRESENTING AT A TERTIARY CARE CENTRE IN KUMAON REGION OF UTRRAKHAND

INTRODUCTION: Macrocytosis is common in various clinical settings and it is found in approximately 1.7– 3.6% of people admitted for care for any cause [1, 2, 3]. Macrocytic anemia is generally classified as megaloblastic or nonmegaloblastic anemia. The causes of macrocytosis fall into two groups: (a) deficiency of vitamin B12 (cobalamin) or folate (or rarely abnormalities of their metabolism) in which the bone marrow is megaloblastic and (b) other causes,in which the bone marrow is usually normoblastic.A high level of suspicion,proper elicitation of the history and thorough examination and investigation of the patient helps in the diagnosis of macrocytic anemia. AIM AND OBJECTIVES: To evaluate the etiology of macrocytic anemia at a tertiary care centre. To determine the etiology of macrocytic anemia.To evaluate clinical manifestations associated with macrocytic anemia. MATERIAL AND METHOD: This 21 month Cross sectional observational study was carried out in OPD/IPD Department of Medicine, Government Medical College Haldwani (Uttarakhand). Full clinical examination and information regarding alcohol intake, dietary habit, drug intake, thyroid disorder and other comorbid illnesses was obtained.All patients were investigated with a completeHaemogramLiver function tests Serum TSH fasting vitamin B12 was measured Ultrasound as and when required. RESULT: In this study106 patients were taken 58 (45.3% ) were male and 48(54.7%) were females. Mean age was 44.83+16.85 years.Hemoglobin and MCV was in the range of 6.42 ± 2.09,(108.24 ± 7.10) respectively .The majority of patient,28 (52.83%) had vit B12 level in the range of 101 – 200 pg/ml,The majority of patients,44 (41.1%) had LDH level in the range of 281 – 1000 IU/L . CONCLUSION: In this study, there was a preponderance of young people.Vegetarians were most susceptible to MA especially cobalamin deficiency. Nutritional deficiency was the most common cause of MA, followed by alcohol and alcoholic liver disease.Data regarding the magnitude of the problem in different parts of India and the factors that might influence its incidence were lacking. Macrocytic anemia must be considered in the differential diagnosis of patients presenting with pyrexia of unknown origin,mild icterus or pancytopenia.

Study of Spectrum of anemia in various age groups

: Anemia is major health problem world-wide especially in developing countries. Globally 1.62 billion persons are affected. It has grave consequences on human health. The present study evaluates the severity and morphology of anemia in various age groups in rural population. : 1): To study degree and severity of anemia in study population; 2): To study distribution of anemia in various age groups; 3): To study morphological spectrum of anemia in study population. s: The study includes 792 patients having low concentration of hemoglobin for their age & sex. The other hematological parameters and morphology were analyzed. : In our study, females constituted 72.6% (575/792) of study population and male constituted 27.4% (217/792) of population. Pediatric patients (up to 15 years of age) constituted 10% of study population. Out of 792 anemic patients, 439 (55.4%) were having moderate anemia, 228 (28.7%) were having mild anemia & 125 (15.78%) were having severe anemia. Out of 792 anemic patients, 384 (48.4%) were having microcytic hypochromic anemia, 296 (37.37%) were having normocytic normochromic anemia, 84 (10.6%) were having macrocytic anemia and 3.5% were having hemolytic anemia. Out of 792 anemic patients, 439 (55.4%) were having moderate anemia, 228 (28.7%) were having mild anemia & 125 (15.78%) were having severe anemia. In our study anemia was more common in females as compared to males constituting majority of study population which is in concordance with other studies also. Moderate anemia was more common in study population as well as in various sub-groups being in concordance with other studies too. Morphologically microcytic hypochromic anemia was the most common type of anemia especially in adult females. Pregnant females were found to have normocytic normochromic anemia predominantly. Most of the cases of hemolytic anemia was found in children <15years. Similarly macrocytic anemia was found more commonly in adult males. Anemia is the most hematological abnormality found in daily practice, though found in all age groups but quite higher in females. The main objective for diagnosing anemia is to make clinician aware so that they can take measures to prevent and control anemia.

A new murine Rpl5(uL18) mutation provides a unique model of variably penetrant Diamond Blackfan Anemia

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins (RPs) are found in the majority of individuals with Diamond Blackfan anemia (DBA) while somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human DBA patients. Here we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations and increased mortality. Following genetic mapping and whole exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ mutant animals had a profound delay in erythroid maturation and increased mortality at embryonic day E12.5, which improved by E14.5. Surviving mutant animals had a macrocytic anemia at birth as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus mutant mouse will be useful to study the factors influencing the variable penetrance that is observed in DBA.

Benign and malignant hematologic manifestations in patients with VEXAS syndrome due to somatic mutations in UBA1

Somatic mutations in UBA1 involving hematopoietic stem and myeloid cells have been reported in patients with the newly defined VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Here, we report clinical hematologic manifestations and unique bone marrow (BM) features in 16 patients with VEXAS. All patients were male and had a history of severe autoinflammatory and rheumatologic manifestations and a somatic UBA1 mutation (p.Met41). Ten patients had hematologic disorders: myelodysplastic syndrome (MDS; 6 of 16), multiple myeloma (2 of 16), monoclonal gammopathy of undetermined significance (2 of 16), and monoclonal B-cell lymphocytosis (2 of 16), and a few of those patients had 2 co-existing clonal processes. Although macrocytic anemia (100%) and lymphopenia (80%) were prevalent in all patients with VEXAS, thrombocytopenia and neutropenia were more common in patients with progression to MDS. All BMs in VEXAS patients had prominent cytoplasmic vacuoles in myeloid and erythroid precursors. In addition, most BMs were hypercellular with myeloid hyperplasia, erythroid hypoplasia, and varying degrees of dysplasia. All patients diagnosed with MDS were lower risk (low blast count, very good to intermediate cytogenetics) according to standard prognostic scoring with no known progression to leukemia. In addition, 10 of 16 patients had thrombotic events, including venous thromboembolism and arterial stroke. Although VEXAS presents symptomatically as a rheumatologic disease, morbidity and mortality are associated with progression to hematologic disease. Given the increased risk of developing MDS and multiple myeloma, surveillance for disease progression is important.

A prospective clinico-etiological study of 100 cases of pancytopenia in a tertiary care hospital in Eastern India

Pancytopenia is a hematological entity which is relatively common. Its evaluation is important for arriving at an early and correct diagnosis, to aid in appropriate management.It was an observational study conducted in the Department of General Medicine and Clinical Hematology of our institute from June 2019 to May 2020. Clinical, hematological and biochemical parameters of 100 pancytopenic patients were evaluated and descriptive statistics was used. The age ranged from 18-75 years. The commonest presentation was easy fatigability and fever. Besides pallor, splenomegaly and hepatomegaly were the presenting signs. Megaloblastic anaemia was detected in 64% followed by aplastic anaemia in 12% and acute leukemia in 6% patients. Among infective etiologies, two cases of malaria (P. falciparum). And each case of HIV, tuberculosis and dengue were seen. Lowest Hb% of 1.8 gm/dl, lowest total leucocyte count (TLC) of 500cells/cmm and lowest total platelet count (TPC) of 4000 cells/cmm was noted in a case of aplastic anemia. Macrocytic anemia was predominant blood picture. Hypercellular marrow was noted in 70(70%) cases and common cause was megaloblastic anemia, followed by leukaemia. Hypocellular marrow was noted in 12(12%) patients with aplastic anemia being commonest cause. In our study diagnosis of pancytopenia and its causes were ascertained by hematological investigations. An early and prompt treatment was given according to the cause and severity. Most of the cases had good prognosis due to a treatable cause.