Spectrum of Genetic Defects and Phenotype-Genotype Correlation in Dyserythropoietic Anemias: Bench to Bedside Approach in the Indian Scenario

Introduction

Congenital dyserythropoietic anemias (CDA) are rare inherited red cell disorders characterized by ineffective erythropoiesis and inappropriate reticulocytopenia. CDAs are usually difficult to diagnose due to variable phenotypes and overlapping bone marrow (BM) morphology with other disorders. Numerous implicated causal genes make Sanger sequencing a less likely approach and hence, the use of targeted resequencing can expedite molecular diagnosis. This study aimed at determining the genetic spectrum of CDAs and translating the results into patient care.

Methods

Twenty nine patients with clinical and laboratory evidence suggestive of CDA and 1 patient suggestive of CDA with thrombocytopenia by BM morphology were studied. Various biochemical and molecular tests were done to exclude common hemolytic anemias. Common SEC23B: p.Tyr462Cys variant in our patients with CDA was screened by Sanger sequencing. DNA libraries were prepared using TruSight One Sequencing Panel and TruSeq Custom Amplicon Panel and sequenced on Illumina platform. After data analysis variants were classified and the most likely disease-causing variants were validated by Sanger sequencing followed by pedigree analysis.

Results

Out of 27 patients of suspected CDA, SEC23B: p.Tyr462Cys variant was found in 10 patients. Rest of the remaining 17 patients were subjected to targeted resequencing. Data analysis revealed novel potentially pathogenic variants in compound heterozygosity in SEC23B in 4 patients and 1 patient had a heterozygous variant in SEC23B. There could be the possibility of intronic or large indel in her. The variants were distributed throughout the SEC23B gene. Notably, in 7 patients with suspected CDA, the final molecular diagnosis were hemolytic anemias. Of them, 4 showed likely pathogenic variants in PKLR gene and 1 each had probably causal variant in MTRR, SPTB and PIEZO1 genes. In the patient's with pyruvate kinase deficiency, screening by enzyme assays were normal. Except for the patient with MTRR gene defect all 6 had transfusion dependent anemia and BM showed dyserythropoiesis. One patient each of GATA1 gene variant (novel) and a known pathogenic variant p.Glu325Lys in KLF1 gene (CDA type IV) was detected. Of 17 cases subjected to targeted resequencing the diagnosis was achieved in ~76% (13/17) of cases.

The phenotypes correlated with the genetic defects found in the SEC23B gene. The homozygous and compound heterozygous defects in this gene cause CDA type II. As anticipated GATA1 gene defect (p.Val205Leu) was found in a patient of X-linked thrombocytopenia with dyserythropoietic anemia. Patient with KLF1 had high levels of fetal hemoglobin along with features of dyserythropoiesis in BM compatible with the phenotype of variant p.Glu325Lys causing CDA type IV. Phenotype-genotype correlation was discrepant in 7 cases of CDA. In 4 cases pyruvate kinase deficiency (PKLR) was found and each case of hereditary xerocytosis (PIEZO1), membrane defect (SPTB) and MTRR defect was found.

Conclusion(s)

CDA showed a highly varied etiology. Our experience demonstrates a high diagnostic yield (~76%) of targeted resequencing for molecular diagnosis of suspected CDAs. Discrepancy was noted in 41% (7/17) cases with suspected CDA which were diagnosed as hemolytic anemia after molecular analysis. Establishing the correct diagnosis of pyruvate kinase deficiency led to an evidence-based decision of splenectomy that eliminated transfusion dependence. In the patient with MTRR defect change in therapy was suggested. Prenatal diagnosis was done for 2 families, where in 1 of the family both the SEC23B variants were novel and in compound heterozygosity. This study highlights the importance of genetic testing in patients under frequent blood transfusions and suspected CDAs, to provide accurate diagnosis and therapeutic interventions.