Congenital dyserythropoietic anemias (CDA) are a rare and heterogeneous group of disorders. CDAs are characterized by ineffective erythropoiesis, morphological abnormalities of erythroblasts (bi/ multinucleated erythroblast, internuclear bridges, erythroid hyperplasia), haemolysis, and hypoglycosylation of red blood cell membrane proteins. Depending on erythroid maturation and precursors involved, three major types of CDA (I, II, III) are identified. Other variants of CDA like transcription factor-related CDA include CDA type IV and X-linked thrombocytopenia with or without dyserythropoietic anemia (XLTDA). The CDA associated with Majeed syndrome is also known. The causative genes are CDAN1, C15ORF41(CDIN2), SEC23B, KIF23, KLF1, GATA1, and LPIN2 .The Patients suffering from CDA are characterized with anemia of variable degree, pallor, recurrent jaundice, hepatomegaly, splenomegaly, gall stones. In some cases, patients are transfusion-dependent, while in some cases, transfusion is less frequently required, and patients are asymptomatic . Light and electron microscopic analysis of bone marrow samples , SDS-PAGE exclusively for CDA type II, and genetic analysis using Sanger sequencing and next-generation sequencing is currently used for the diagnosis of CDA.
Congenital Dyserythropoietic Anemia type II (OMIM: 224100) is the most common type of CDA. The genetic defect of CDA type II is due to mutations in the SEC23B gene (encoding COPII) . SEC23B gene (OMIM: 610512) is located on chromosome 20p11.23. It is involved in providing instructions for the making of one component of coat protein complex II (COPII). COPII is a large group of interacting proteins that function in the formation of vesicles. Vesicles are small sac-like structures that are involved in the transportation of proteins and other materials in cells. COPII triggers the formation of vesicles in the endoplasmic reticulum (ER). It plays a vital role in protein processing and transportation. Proteins that are destined to be secreted are carried by COPII vesicles. Endoplasmic reticulum (ER)-to-Golgi trafficking is disturbed due to abnormalities in the SEC23B gene. This affects different glycosylation pathways and ultimately accounts for the cellular phenotype observed in CDAII .
SEC23B mutations are inherited in an autosomal recessive pattern. So far, only 16 patients of congenital dyserythropoietic anemia type II from India have been described in the literature: 6 of the patients diagnosed based on bone marrow light microscopy , 10 North Indian patients having Y462C mutation in exon 12 of SEC23B gene [7–8]. There is a possibility of more patients suffering from CDA in the Indian population. But, the data is not available because of difficulties in diagnosis. Biochemical methods like the anti-CD44 antibody binding test, along with bone marrow microscopy and molecular tests, will be helpful [9–10]. More than 120 mutations have been identified in the SEC23B gene that causes CDA Type II. The most common mutation in the Indian population is c.1385A>G, p.Y462C . Many patients with CDA remain undiagnosed because of a lack of simple, confirmatory diagnostic methods.
In the Eosin-5’-Maleimide assay (EMA), patients suffering from hereditary spherocytosis (HS) and CDA type II show decreased mean channel fluorescence (MCF). Thus, many cases of CDA type II are misdiagnosed as cases of HS. In CDA type II patients, anti-CD44 antibody binding to red blood cells is raised compared to normal healthy individuals and HS patients. We used the Eosin-5’-Maleimide assay (EMA) by flow cytometry to diagnose hereditary spherocytosis and CDA type II. We have further used an anti-CD44 antibody binding assay to suspect the diagnosis of CDA II in patients who have shown low mean channel fluorescence in the EMA test.
High-resolution melting (HRM) curve analysis is a polymerase chain reaction (PCR) based technique applied to identify genetic differences and scan nucleic acid sequences. The properties of DNA such as length, sequence, composition, GC content, and heterozygosity are responsible for the melting curve. The significant information about the genotype can be obtained by these melting curve profiles, which allows an analysis of mutations and polymorphisms. The precision and accuracy of the HRM method are dependent on the fluorescent dye used along with the instrument and software in the analysis . HRM analysis gives reproducible and rapid results though it is not used in routine clinical practice . In this study, we have developed the HRM method to detect common SEC23Bgene mutation causing CDA type II in the Indian population.