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.
Anemia ya kuzaliwa (CDA) ni aina ya ugonjwa wa kawaida wa ugonjwa wa kuzaliwa. CDAs ni sifa ya erythropoiesis ineffective, morphological abnormalities ya erythroblasts (bi- multinucleated erythroblast, internucleated bridges, erythropoietic hyperplasia), hemolysis, na hypoglycosylation ya nyekundu ya damu cell membrane protini. Kulingana na kukomaa kwa erythroid na vichwa vya habari vinavyohusika, aina kuu tatu za CDA (I, II, III) zimetambuliwa. CDA ya aina ya IV (CDA ya aina ya IV) na X-linked thrombocytopenia (XLTDA) CDA inayohusiana na ugonjwa wa Majeed pia inajulikana. CDA (C15-ORF41) (CDIN-2), C15-ORF2 (CDIN-2), C15-ORF2 (CDIN-2), C15-ORF2 (CDIN-2), C15-ORF2 (CDIN-2), C15-ORF2 (CDIN-2), C16-ORF2 (CDIN-2), C16-ORF2 (CDIN-3) na C16-ORF2 (CDIN-3) ni aina ya ugonjwa unaosababishwa na ugonjwa wa upungu wa damu (anemia) na ugonjwa wa gallstones (gallstones). Katika baadhi ya kesi, wagonjwa wanategemea uingizaji damu, wakati katika baadhi ya kesi, uingizaji damu huhitajika mara chache, na wagonjwa hawana dalili. Uchunguzi wa microscopic na electron ya sampuli za mifupa ya mifupa, SDS-PAGE, na uchambuzi wa maumbile kwa kutumia utaratibu wa Sanger na utaratibu wa kizazi kijacho, hutumiwa kwa sasa kwa utambuzi wa CDA. Anemia ya congenital dyserythropoietic aina ya II (OMIM 224100) ni aina ya kawaida ya CDA. Ugonjwa wa CDA2 unawezekana kutokana na mabadiliko katika jeni la SEC23B (COPD-2). Jeni la SEM23B (OMIM 61012), ni sehemu ya chromosome 20p1123 Inafanya kazi katika kuandaa maagizo ya kutengeneza sehemu moja ya coat protein complex II (COPII). CO2 ni kundi kubwa la protini zinazohusika katika kuunda vesicles. Vesicles ni mifumo midogo kama mifuko ambayo inahusika katika usafirishaji wa protini na vifaa vingine katika seli. COPD huchochea uundaji wa vesicles katika reticulum ya endoplasmic (ER). Ina jukumu muhimu katika usindikaji na usafirishaji wa protini. Protini ambazo zimekusudiwa kutolewa hubebwa na vesicles za COPII. Endoplasmic reticulum (ER) - Golgi trafiki ni kusumbuliwa kutokana na kasoro katika jeni SEC23B. Hii huathiri njia tofauti za glycosylation na hatimaye huelezea phenotype ya seli iliyoonekana katika CDAII. Mabadiliko ya SEC23B ni urithi katika mtindo wa autosomal recessive. Kwa sasa, wagonjwa 16 tu wa aina ya 2 ya anemia ya dyserythropoietic ya kuzaliwa kutoka India wameelezwa katika fasihi: 6 ya wagonjwa waliogunduliwa kulingana na microscopy ya mwanga ya uti wa mgongo, 10 wagonjwa wa India Kaskazini wenye mabadiliko ya Y462C katika exon 12 ya jeni la 23SEB. Kuna uwezekano wa wagonjwa zaidi wanaoteseka kutokana na CDA katika idadi ya watu wa India. Lakini, data haipatikani kwa sababu ya matatizo katika utambuzi. Kwa mfano, kwa mfano, mtihani wa anti-CD44 au mtihani wa kuunganisha antibody, pamoja na uchunguzi wa microscopy ya mifupa na vipimo vya molekuli, inaweza kusaidia. Mabadiliko zaidi ya 120 yamegunduliwa katika jeni la SEC23B ambalo husababisha CDA ya Aina ya II. Mabadiliko ya kawaida katika idadi ya watu wa India ni c1385A>G, pY462C. Wagonjwa wengi wenye CDA hawajatambuliwa kwa sababu ya ukosefu wa mbinu rahisi za uthibitisho wa utambuzi. Katika Eosine-5-Maleimide Assay (EMA), wagonjwa wanaosumbuliwa na spherocytosis ya urithi (HS) na CDA aina ya II huonyesha kupungua kwa fluorescence ya kituo cha wastani (MCF). Kwa hiyo, visa vingi vya CDA aina ya pili huchunguzwa kimakosa kuwa visa vya HS. Katika wagonjwa wa CDA ya aina ya II, antibody ya anti-CD44 inayounganisha kwa seli nyekundu za damu imeongezeka ikilinganishwa na watu wazima wa kawaida na wagonjwa wa HS. Eosine-5-Maleimide (EMA) ni kipimo cha kawaida cha mtiririko wa cytometry, ambacho kinachotumiwa kugundua spherocytosis ya urithi na CDA ya aina ya 2. Kwa mfano, kwa ajili ya uchunguzi wa antibody-binding ya anti-CD44, CDA II iligunduliwa kwa wagonjwa ambao walikuwa na fluorescence ya chini ya kituo cha wastani katika mtihani wa EMA. High-resolution melting curve analysis (HRM) ni mbinu ya polymerase chain reaction (PCR) inayotumika kutambua tofauti za maumbile na kuchunguza mfululizo wa asidi ya nuklia. Sifa za DNA kama vile urefu, mfululizo, muundo, maudhui ya GC, na heterozygosity ni wajibu kwa ajili ya mteremko wa kuyeyuka. Habari muhimu kuhusu genotype inaweza kupatikana na hizi profile kuyeyushwa curve, ambayo inaruhusu uchambuzi wa mutations na polymorphisms. Uwezo wa utaratibu wa HRM unategemea rangi ya fluorescent iliyotumiwa pamoja na chombo na programu katika uchambuzi. 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