Erythroid Krüppel-Like Factor (KLF1): A Surprisingly Versatile Regulator of Erythroid Differentiation.

Erythroid Krüppel-like factor (KLF1), first discovered in 1992, is an erythroid-restricted transcription factor (TF) that is essential for terminal differentiation of erythroid progenitors. At face value, KLF1 is a rather inconspicuous member of the 26-strong SP/KLF TF family. However, 30 years of research have revealed that KLF1 is a jack of all trades in the molecular control of erythropoiesis. Initially described as a one-trick pony required for high-level transcription of the adult HBB gene, we now know that it orchestrates the entire erythroid differentiation program. It does so not only as an activator but also as a repressor. In addition, KLF1 was the first TF shown to be directly involved in enhancer/promoter loop formation. KLF1 variants underlie a wide range of erythroid phenotypes in the human population, varying from very mild conditions such as hereditary persistence of fetal hemoglobin and the In(Lu) blood type in the case of haploinsufficiency, to much more serious non-spherocytic hemolytic anemias in the case of compound heterozygosity, to dominant congenital dyserythropoietic anemia type IV invariably caused by a de novo variant in a highly conserved amino acid in the KLF1 DNA-binding domain. In this chapter, we present an overview of the past and present of KLF1 research and discuss the significance of human KLF1 variants.

Identification of a novel splice variant in SEC23B gene in a patient with concomitant presence of congenital dyserythropoietic anemia II and Gilbert's syndrome.

BACKGROUND: Congenital dyserythropoietic anemia Ⅱ (CDA Ⅱ) is a rare inherited disorder of defective erythropoiesis caused by SEC23B gene mutation. CDA Ⅱ is often misdiagnosed as a more common type of clinically related anemia, or it remains undiagnosed due to phenotypic variability caused by the coexistence of inherited liver diseases, including Gilbert's syndrome (GS) and hereditary hemochromatosis. METHODS: We describe the case of a boy with genetically undetermined severe hemolytic anemia, hepatosplenomegaly, and gallstones whose diagnosis was achieved by targeted next generation sequencing. RESULTS: Molecular analysis revealed a maternally inherited novel intronic variant and a paternally inherited missense variant, c.[994-3C > T];[1831C > T] in the SEC23B gene, confirming diagnosis of CDA Ⅱ. cDNA analysis verified that the splice acceptor site variant results in two mutant transcripts, one with an exon 9 skip and one in which exons 9 and 10 are deleted. SEC23B mRNA levels in the patient were lower than those in healthy controls. The patient was also homozygous for the UGT1A1*6 allele, consistent with GS. CONCLUSION: Identification of the novel splice variant in this study further expands the spectrum of known SEC23B gene mutations. Molecular genetic approaches can lead to accurate diagnosis and management of CDA Ⅱ patients, particularly for those with GS coexisting.

Congenital dyserythropoietic anemia type II in a newborn with a novel compound heterozygous mutation in the SEC23B: a case report and review of the literature.

Congenital dyserythropoietic anemia type II (CDA II) refers to a group of extremely rare heterozygous disorders characterized by ineffective erythropoiesis and morphological abnormalities of erythrocytes and bone marrow erythroblasts. Six types of CDA with differing heterogenous genetic mutations have been identified to date. Due to the genetic and clinical heterogeneity of CDA, accurate diagnosis can be very challenging, especially with the clinical overlap observed between CDA and other dyserythropoietic diseases. A 1-month-old infant girl, born to a non-consanguineous family, presented with severe normocytic anemia that required transfusions every 2 to 3 weeks since birth, as well as jaundice. Whole exome sequencing revealed a novel compound heterozygosity in the SEC23B gene, thus establishing the diagnosis of CDA II. Analysis by multiple bioinformatics tools predicted that the mutant proteins were deleterious. Here, we report a novel variation in SEC23B that extends the mutation spectrum of SEC23B in the diagnosis of CDA II.

Congenital Dyserythropoietic Anemia Type I: A Rare Case Report.

Congenital dyserythropoietic anemias are a group of rare hereditary conditions affecting erythropoiesis. These disorders are characterized by anemia, primarily caused by inefficient erythropoiesis, as well as distinctive morphological abnormalities observed in most erythroblasts in the bone marrow. congenital dyserythropoietic anemia type I (CDA-I) is a hereditary condition characterized by inefficient production of red blood cells and excessive accumulation of iron. It follows an autosomal recessive pattern of inheritance. There have been approximately 300 recorded cases of CDA-I documented on a global scale. CDA-I is a rarely documented condition in the Indian subcontinent. Therefore, we will be examining a case of CDA-I in the present article. A male infant, aged four months, who had signs of vomiting, weight loss, and failure to thrive, was diagnosed with CDA-I following a bone marrow aspiration. Our experience provides further evidence supporting the notion that the accurate diagnosis of CDA-I can be achieved by doing a comprehensive assessment of bone marrow aspiration.

Development of High-Resolution Melting Curve Analysis for rapid detection of SEC23B gene mutation causing Congenital Dyserythropoietic Anemia type II in Indian population.

BACKGROUND: Congenital dyserythropoietic anemias (CDAs) are a very rare and heterogeneous group of disorders characterized by ineffective erythropoiesis. CDA II is caused by mutations in the SEC23B gene. The most common mutation reported in India is c.1385 A > G, p.Y462C. There is no simple and cost-effective confirmatory diagnostic test available for CDA, and therefore, many patients remain undiagnosed. High-resolution melting curve (HRM) analysis is a polymerase chain reaction (PCR) based technique applied to identify genetic differences and scan nucleic acid sequences. HRM can be used to rapidly screen the common mutation causing CDA II in the Indian population. Thus, we studied the use of High-Resolution Melting Curve Analysis to detect common mutation causing CDA II in the Indian population. METHOD: 11 patients having SEC23B (Y462C) mutation causing CDA II are considered for this study. HRM was used to check the presence of Y462C mutation. To verify the accuracy of the HRM analysis, we compared HRM results with the results of Sanger sequencing. This helped us to confirm the diagnosis. RESULTS: We have described the clinical, hematological, and genetic data of eleven patients suffering from CDAII. According to HRM and Sanger sequencing, a homozygous SEC23B (Y462C) mutation was present in all patients, whereas a heterozygous Y462C mutation was present in their parents. CONCLUSION: Our data showed that High-Resolution Melting (HRM) analysis could be used to rapidly screen common SEC23B mutation that causes CDA II in the Indian population.

New Cases and Mutations in SEC23B Gene Causing Congenital Dyserythropoietic Anemia Type II.

Congenital dyserythropoietic anemia type II (CDA II) is an inherited autosomal recessive blood disorder which belongs to the wide group of ineffective erythropoiesis conditions. It is characterized by mild to severe normocytic anemia, jaundice, and splenomegaly owing to the hemolytic component. This often leads to liver iron overload and gallstones. CDA II is caused by biallelic mutations in the SEC23B gene. In this study, we report 9 new CDA II cases and identify 16 pathogenic variants, 6 of which are novel. The newly reported variants in SEC23B include three missenses (p.Thr445Arg, p.Tyr579Cys, and p.Arg701His), one frameshift (p.Asp693GlyfsTer2), and two splicing variants (c.1512-2A>G, and the complex intronic variant c.1512-3delinsTT linked to c.1512-16_1512-7delACTCTGGAAT in the same allele). Computational analyses of the missense variants indicated a loss of key residue interactions within the beta sheet and the helical and gelsolin domains, respectively. Analysis of SEC23B protein levels done in patient-derived lymphoblastoid cell lines (LCLs) showed a significant decrease in SEC23B protein expression, in the absence of SEC23A compensation. Reduced SEC23B mRNA expression was only detected in two probands carrying nonsense and frameshift variants; the remaining patients showed either higher gene expression levels or no expression changes at all. The skipping of exons 13 and 14 in the newly reported complex variant c.1512-3delinsTT/c.1512-16_1512-7delACTCTGGAAT results in a shorter protein isoform, as assessed by RT-PCR followed by Sanger sequencing. In this work, we summarize a comprehensive spectrum of SEC23B variants, describe nine new CDA II cases accounting for six previously unreported variants, and discuss innovative therapeutic approaches for CDA II.

Rare congenital Dyserythropoietic anemia of childhood: A case report.

Congenital dyserythropoietic anemias (CDA) is a heterogeneous class of anemia of varying degrees of ineffective erythropoiesis and secondary hemochromatosis. We reported a case of CDA and showed our approach to reaching a diagnosis, highlighting the importance of the typical morphological appearance of bone marrow erythroblasts to reach the diagnosis.

Ultrastructural characteristics of erythroid cells in congenital dyserythropoietic anemia type II, with a focus on peripheral cisternae and double membranes.

Peripheral cisternae and double membranes (PCDMs) in erythroid cells are a landmark of type II congenital dyserythropoietic anemia (CDA). To gain further insights into the mechanism of dyserythropoiesis, erythroblasts and erythrocytes in bone marrow were studied in 22 Chinese patients with CDA Ⅱ by transmission electron microscopy. The study demonstrated an increase in all patients in erythroblasts with PCDMs with development from pro-erythroblast to red blood cells. PCDMs often connected with cisternae of endoplasmic reticulum (ER) and the perinuclear space, and were accompanied by karyopyknosis, karyolysis and disruption in polychromatic and orthochromatic erythroblasts. The results suggest that PCDMs are transformed from ER during erythropoiesis and participate in the dissolution and deletion of late erythroid cells in patients with CDA II.

Pyruvate kinase deficiency mimicking congenital dyserythropoietic anemia type I.

BACKGROUND: Pyruvate kinase (PK) deficiency is the most common enzyme abnormality in the glycolytic pathway. Here, we describe two siblings with PK deficiency that mimicked congenital dyserythropoietic anemia (CDA) type I. CASE: The siblings were referred to our hospital for evaluation of anemia when they were newborns. Their PK enzyme activities were normal. Their bone marrow aspirations and electron microscopies showed CDA-like findings. A CDA panel with next-generation sequencing showed no mutation. Though their PK enzyme levels were normal, a molecular study of the PKLR gene showed a homozygous variant c.1623G > C (p.Lys541Asn) in exon 12 of our patients. CONCLUSIONS: Although the diagnosis of pyruvate kinase deficiency is difficult, it can be confused with many other diagnoses. Bone marrow findings of these cases are similar to congenital dyserythropoietic anemia. In patients with normal pyruvate kinase enzyme levels, the diagnosis cannot be excluded and genetic analysis is required.

Congenital Dyserythropoietic Anemia Type II: A Case Report.

Congenital dyserythropoietic anemia (CDA) type 2 is a rare genetic disease that presents with mild to severe anemia. The rare occurrence may be a reason why CDAs are often misdiagnosed since the morphological abnormalities and the clinical features are commonly found in other clinically-related anemias. We report a case of a 17-year-old male who presented in a tertiary care government hospital, with a history of lethargy, abdominal pain, abdominal fullness, and failure to thrive. Bone marrow biopsy reported the uncommon diagnosis of CDA type 2, the Ham test was also positive. The management included a multi-disciplinary approach alongside counseling of the family.

[Differential diagnosis of inherited bone marrow failure syndromes in erythrocyte disorders].

Diamond-Blackfan anemia (DBA), congenital dyserythropoietic anemia (CDA), and inherited sideroblastic anemia (ISA) are representative diseases of inherited bone marrow failure syndromes in erythrocyte diseases. DBA is primarily caused due to ribosomal dysfunctions. Furthermore, reticulocytes and erythroid progenitor cells decrease considerably within the peripheral blood and bone marrow, respectively. CDA is caused by a disturbance in red blood cell maturation and ineffective erythropoiesis due to hemolysis in the bone marrow. CDA is mainly classified into types I to III, and multinucleated erythroblasts observed in the bone marrow, typically in the internuclear bridge in type I. ISA is caused by iron metabolism dysfunction in the mitochondria due to defective heme synthesis. Sideroblasts appear ringed due to iron accumulation in the mitochondria of erythroid precursors. Gene mutation analysis is indispensable for the confirmatory diagnosis of these diseases; however, narrowing down the diagnosis, by examining the erythrocytes in the peripheral blood and the erythroblast morphology in the bone marrow, is also important.

Ablation of Tmcc2 Gene Impairs Erythropoiesis in Mice.

(1) Background: Transcriptomic and proteomic studies provide a wealth of new genes potentially involved in red blood cell (RBC) maturation or implicated in the pathogenesis of anemias, necessitating validation of candidate genes in vivo; (2) Methods: We inactivated one such candidate, transmembrane and coiled-coil domain 2 (Tmcc2) in mice, and analyzed the erythropoietic phenotype by light microscopy, transmission electron microscopy (TEM), and flow cytometry of erythrocytes and erythroid precursors; (3) Results: Tmcc2-/- pups presented pallor and reduced body weight due to the profound neonatal macrocytic anemia with numerous nucleated RBCs (nRBCs) and occasional multinucleated RBCs. Tmcc2-/- nRBCs had cytoplasmic intrusions into the nucleus and double membranes. Significantly fewer erythroid cells were enucleated. Adult knockouts were normocytic, mildly polycythemic, with active extramedullary erythropoiesis in the spleen. Altered relative content of different stage CD71+TER119+ erythroid precursors in the bone marrow indicated a severe defect of erythroid maturation at the polychromatic to orthochromatic transition stage; (4) Conclusions: Tmcc2 is required for normal erythropoiesis in mice. While several phenotypic features resemble congenital dyserythropoietic anemias (CDA) types II, III, and IV, the involvement of TMCC2 in the pathogenesis of CDA in humans remains to be determined.

Multiple oral and cerebral relapses of a Granular cell tumor (Abrikossoff Tumor) in a young girl affected by congenital dyserythropoietic anemia type II.

CASE REPORT: A 14-year-old girl presented with 1 cm large whitened lesion on the ventral surface of the tongue, appeared from 1 month. Past history showed congenital dyserythropoietic anemia type II. The lesion was excised and microscopic and immunohistochemical analyses were compatible with benign Abrikossoff tumor. Total body MRI was negative. After six months the patient presented a second tongue lesion and four months later another large painful lesion in the soft palate, with the same istological diagnosis. In addition, she had other multiple lesions: two apperead at pharyngeal level (not biopsied) that remain stable over time, and one at the pituitary gland. CONCLUSION: Granular cell tumors, with or without multiple lesions, are rare in children. About 50% of cases involve the head and neck region, with the tongue being the most affected site. Therapy is based on the surgical excision of the lesions; however some tumor forms, although their histological aspect of benignity, often have an important infiltrative power, making the therapeutic approach difficult, as in our case.

Erythroblastic synartesis associated with lymphoproliferative disorder: There can be more than meets the eye.

Erythroblastic synartesis is a rare cause of acquired dyserythropoiesis. Only 9 cases have been previously reported. We hereby report 3 cases of patients diagnosed with erythroblastic synartesis associated with monoclonal immunoglobulin and an overt malignant lymphoid disorder. A different B-cell clone may produce the monoclonal immunoglobulin, forming a biclonal disorder. In light of these data and literature review, treatment targeting the paraprotein seems to be efficient to control synartesis and correct anemia. In the case of monoclonal gammapathy associated with chronic lymphocytic leukemia, therapeutics should be adapted to control both chronic lymphocytic leukemia and monitored monoclonal immunoglobulin titer.

Novel PKLR missense mutation (A300P) causing pyruvate kinase deficiency in an Omani Kindred-PK deficiency masquerading as congenital dyserythropoietic anemia.

We report herein a child with transfusion-dependent chronic anemia, the cause of which was difficult to establish because of his transfusion dependency. The clinical and laboratory features suggested a chronic nonspherocytic hemolytic anemia (CNSHA) with bone marrow features suggestive of congenital dyserythropoietic anemia (CDA). DNA studies, however, revealed the underlying condition to be due to a novel mutation in the PKLR gene responsible for pyruvate kinase deficiency (PKD). Molecular investigations by a targeted next-generation sequencing (t-NGS) using a custom panel of 71 genes involved in the red blood cell (RBC) disorders revealed that the patient was homozygous for a novel missense mutation c.898G>C, p.Ala300Pro, whereas both his parents were heterozygous for the same mutation.

A common human missense mutation of vesicle coat protein SEC23B leads to growth restriction and chronic pancreatitis in mice.

SEC23B is one of two vertebrate paralogs of SEC23, a key component of the coat protein complex II vesicles. Complete deficiency of SEC23B in mice leads to perinatal death caused by massive degeneration of professional secretory tissues. However, functions of SEC23B in postnatal mice and outside professional secretory tissues are unclear. In this study, we generated a Sec23b KO mouse and a knockin (KI) mouse with the E109K mutation, the most common human mutation in congenital dyserythropoietic anemia type II patients. We found that E109K mutation led to decreases in SEC23B levels and protein mislocalization. However, Sec23bki/ki mice showed no obvious abnormalities. Sec23b hemizygosity (Sec23bki/ko) was partially lethal, with only half of expected hemizygous mice surviving past weaning. Surviving Sec23bki/ko mice exhibited exocrine insufficiency, increased endoplasmic reticulum stress and apoptosis in the pancreas, and phenotypes consistent with chronic pancreatitis. Sec23bki/ko mice had mild to moderate anemia without other typical congenital dyserythropoietic anemia type II features, likely resulting from exocrine insufficiency. Moreover, Sec23bki/ko mice exhibited severe growth restriction accompanied by growth hormone (GH) insensitivity, reminiscent of Laron syndrome. Growth restriction is not associated with hepatocyte-specific Sec23b deletion, suggesting a nonliver origin of this phenotype. We propose that inflammation associated with chronic pancreatic deficiency may explain GH insensitivity in Sec23bki/ko mice. Our results reveal a genotype-phenotype correlation in SEC23B deficiency and indicate that pancreatic acinar is most sensitive to SEC23B deficiency in adult mice. The Sec23bki/ko mice provide a novel model of chronic pancreatitis and growth retardation with GH insensitivity.

Majeed Syndrome: Five Cases With Novel Mutations From Unrelated Families in India With a Review of Literature.

OBJECTIVE: Majeed syndrome (MJS) is an autosomal recessive, systemic autoinflammatory disease (SAID) caused by biallelic loss-of-function variants in the LPIN2 gene. It is characterized by early-onset chronic recurrent multifocal osteomyelitis (CRMO), dyserythropoietic anemia, and neutrophilic dermatosis. We analyzed a cohort of uncharacterized Indian patients for pathogenic variants in LPIN2 and other genes associated with SAIDs. METHODS: We performed whole-exome sequencing (WES) for 1 patient and next-generation sequencing (NGS) targeted gene panel for SAIDs in 3 patients. One patient was a referral from neurology after clinical exome sequencing identified a novel variant in LPIN2. We reviewed the literature for all published studies of mutation-positive MJS patients and have summarized their clinical features and disease-causing variants. RESULTS: We describe the largest series of patients with MJS outside of the Middle East. All 5 patients are homozygous for novel, possibly pathogenic variants in the LPIN2 gene. Two of these variants are missense substitutions, and 3 are predicted to alter transcript splicing and create a truncated protein. In addition to the classical features of CRMO and anemia, patients exhibited previously unreported features, including abdominal pain, recurrent diarrhea/ear discharge, and erythema nodosum. CONCLUSION: Patients with MJS may present initially to different specialists, and thus it is important to create awareness in the medical community. In India, consanguinity is a common sociocultural factor in many ethnic communities and an abbreviated NGS gene panel for autoinflammatory diseases should include MJS. The unavailability of interleukin 1 inhibitors in some countries poses a treatment challenge.

Congenital dyserythropoietic anemia type I: First report from the Congenital Dyserythropoietic Anemia Registry of North America (CDAR).

Congenital dyserythropoietic anemias (CDAs) are characterized by ineffective erythropoiesis and distinctive erythroblast abnormalities; the diagnosis is often missed or delayed due to significant phenotypic heterogeneity. We established the CDA Registry of North America (CDAR) to study the natural history of CDA and create a biorepository to investigate the pathobiology of this heterogeneous disease. Seven of 47 patients enrolled so far in CDAR have CDA-I due to biallelic CDAN1 mutations. They all presented with perinatal anemia and required transfusions during infancy. Anemia spontaneously improved during infancy in three patients; two became transfusion-independent rapidly after starting interferon-α2; and two remain transfusion-dependent at last follow-up at ages 5 and 30 y.o. One of the transfusion-dependent patients underwent splenectomy at 11 y.o due to misdiagnosis and returned to medical attention at 27 y.o with severe hemolytic anemia and pulmonary hypertension. All patients developed iron overload even without transfusions; four were treated with chelation. Genetic testing allowed for more rapid and accurate diagnosis; the median age of confirmed diagnosis in our cohort was 3 y.o compared to 17.3 y.o historically. In conclusion, CDAR provides an organized research network for multidisciplinary clinical and research collaboration to conduct natural history and biologic studies in CDA.

[Whole exome sequencing analysis of compound heterozygous variants of CDAN1 gene in a Chinese family with non-immune hydrops fetalis].

OBJECTIVE: To study the clinical characteristics and genetic variants in a family with non-immune hydrops fetalis. METHODS: Peripheral blood samples were collected from a pregnant woman with suspected non-immune hydrops fetalis of the fetus for routine blood analysis, Rh typing and TORCH test. Amniotic fluid sample was collected for G-banded chromosomal karyotyping. The genomic DNA of the proband was extracted for analysis of chromosomal abnormalities using copy number variation sequencing. Whole-exome sequencing (Trios-WES) was performed on Illumina NovaSeq 6000 platform and exonic DNA was enriched using Agilent Sure Select XT Human All Exon V6. Sorting intolerant from tolerant (SIFT), I-mutant2, PolyPhen-2 and PROVEAN were used to predict the potential effects of amino acid substitution on protein function and splicing variation. The spatial structure of codanin-1 was modeled and visualized with Alpha Fold 2 and PyMOL 2.3 software, and the variants with potential clinical significance were confirmed by Sanger sequencing. RESULTS: Fetal ultrasound at 17 weeks of gestation showed extensive subcutaneous edema, ascites, pleural effusion, enlarged liver and spleen, thickened placenta and pericardium defect. NGS reveals that proband has carried c.2140C>T, p.R714W, and c.1264_1265delCT, p.L422* compound heterozygous variants of CDAN1 gene, which were found to be pathogenic and inherited from proband's father and mother respectively. CONCLUSION: We identified a novel heterozygous CDAN1 gene mutation causing fetal-onset congenital dyserythropoietic anemia type 1, which triggers non-immune hydrops fetalis.

Severe anemia caused by dominant mutations in Krüppel-like factor 1 (KLF1).

The etiology and severity of anemia, a common blood disorder, are diverse. Dominant mutations in Krüppel-like factor 1 (KLF1/EKLF) underlie the molecular basis for some of them. KLF1 is a zinc finger transcription factor that plays an essential role in red blood cell proliferation and differentiation. Mutations have been identified in the KLF1 gene that cause hematologic diseases. Two of these alter one allele but generate an extreme phenotype: the mouse Nan mutation (E339D) leads to hemolytic neonatal anemia with hereditary spherocytosis, and the human CDA mutation (E325K) causes congenital dyserythropoietic anemia (CDA) type IV. These modify functionally important amino acids in the zinc finger DNA-binding domain at positions involved in direct interactions with regulatory elements of KLF1's target genes. Although the two dominant mutations alter the same evolutionarily conserved glutamic acid residue, the substitutions are not equivalent and lead to divergent consequences for the molecular mechanisms underlying activity of these mutants, particularly in recognition and interaction with their unique binding sites. Consequently, the properties of the protein are transformed such that it acquires novel dominant characteristics whose effects may not be limited to the erythroid compartment. KLF1 mutants cause loss-of-function/haploinsufficiency effects on some KLF1 wild-type target genes, while at the same time gain-of-function effects activate ectopic sites and neomorphic gene expression. Such anomalies not only lead to intrinsic red cell problems, but also to expression of non-erythroid genes that systemically disturb organ development. This review highlights recent molecular, biochemical, and genetic studies of KLF1 mutants, particularly the dramatic consequences that come from just a single amino acid change. The study of these variants provides an important contribution to the overall understanding of the DNA-protein interface of the zinc finger subtype of transcription factors, and the potential clinical consequences of what might appear to be a minor change in sequence.