Stepwise genetic testing refines the diagnosis of suspected congenital dyserythropoietic anemias: A cohort study of 76 Indian families

Abstract Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of inherited red cell disorders characterized by ineffective erythropoiesis, and distinctive bone marrow dyserythropoiesis. Diagnostic challenges arise from genetic heterogeneity and overlapping marrow morphology with other inherited hemolytic anemia and bone marrow failure syndromes. Over the period of 15-year, we analyzed 83 individuals from 76 families with suspected CDA to delineate the genetic basis and assess clinical implications of molecular diagnosis. Cases were enrolled following exclusion of common acquired and inherited causes of anemia and hemolysis. Bone marrow in all patients showed significant dyserythropoiesis. Genetic evaluation began with Sanger screening for the SEC23B:p.Tyr462Cys founder variant, followed by next-generation sequencing (NGS) in negative cases. Pathogenic/likely pathogenic variants were confirmed by Sanger sequencing and family studies (where available). The SEC23B:p.Tyr462Cys variant was identified in homozygous form in 35 individuals from 31 families and in heterozygous form in four families using Sanger sequencing. NGS in remaining cases revealed compound heterozygous SEC23B variants in five families and a single heterozygous variant in two families. In six families, only one SEC23B variant was detected, suggesting potential structural variants or intronic splice sites. NGS identified CDA subtypes with genotype phenotype concordance: KLF1-related CDA IV with elevated HbF, RACGAP1-associated CDA IIIb with giant multinucleated erythroblasts, and two cases with GATA1 variants presenting as X-linked thrombocytopenia with dyserythropoiesis. Phenotype-Genotype Discordance: Importantly, 17 cases were reclassified based on genetic findings, including PKLR-related pyruvate kinase deficiency (7 cases), hereditary pyropoikilocytosis (SPTA1, SPTB, 4 cases), PIEZO1-related xerocytosis (2 cases), and single cases of MTRR (Homocystinuria-megaloblastic anemia, cbl E type), HbH disease (HBA2), Diamond-Blackfan anemia (RPS19), and SAMD9L-associated anemia. These reclassified cases had significant management implications. A patient with MTRR defect responded to a change in therapy. Splenectomy in a PKLR-deficient patient led to transfusion independence in all except one where tanfusion requirement was reduced. Iron overload was noted in several CDA II transfusion-independent, requiring chelation. Five families underwent successful prenatal diagnosis. One patient with KLF1-related CDA IV underwent bone marrow transplantation with favorable outcome. This study represents the largest series of genetically characterized CDA from South Asia. It demonstrates the utility of tiered molecular testing, starting with cost-effective Sanger screening for common variant SEC23B:p.Tyr462Cys, and extending to NGS for comprehensive diagnosis. Accurate molecular characterization enabled precise therapy, avoided inappropriate interventions (e.g., splenectomy in stomatocytosis), informed genetic counseling, and facilitated prenatal and curative interventions. Our findings underscore the diverse genetic landscape of suspected CDAs in the Indian population and highlight the pivotal role of integrative diagnostics in optimizing patient care.