The Spectrum of Genetic Defects in Indian Patients with Rare Congenital Anemias: Next Generation Sequencing Based Approach

Introduction Diagnosis of inherited anemias is based on automated red cell indices, morphology and reticulocyte count in an appropriate clinical presentation and a stepwise diagnostic algorithm needs to be followed. Major inherited causes of hemolysis include the hemoglobinopathies, membranopathies and enzymopathies. In contrast, ineffective erythropoiesis, a term that refers to a disturbance in proliferation, differentiation and maturation of erythroblasts, includes megaloblastic anemia, myelodysplastic syndromes, thalassemias, and congenital dyserythropoietic anemias (CDA). Phenotypes vary from severe transfusion-dependent hemolytic anemia (HA) to fully compensated hemolysis. Patients are encountered where the entire spectrum of diagnostic tests cannot identify the etiology. Causal genes implicated are numerous, making a gene-by-gene approach time consuming, expensive and laborious. Hence, the use of targeted resequencing can expedite molecular diagnosis, genetic counseling and prenatal diagnosis if indicated. Objectives This study aimed at determining the spectrum of mutations in uncommon HA and CDA. Methods Sixty-one patients (HA=44, CDA=17) with clinical and laboratory evidence suggestive of HA/CDA were enrolled after excluding common causes of anemias. Various biochemical and molecular tests were used to exclude glucose-6-phosphate dehydrogenase (G6PD) deficiency, hemoglobinopathies, autoimmune HA, hereditary spherocytosis and pyruvate kinase (PK) deficiency. Peripheral blood genomic DNA was extracted using QIAamp DNA Blood Midi Kit, quantified on NanoDrop 2000 spectrophotometer and Qubit® 2.0 Fluorometer. Common G6PD, PKLR variants, SEC23B mutation were excluded by molecular tests. Since the genetic defects in Indian patients with unexplained HA/CDA are not available, we first tested 16 patients with a comprehensive commercially available sequencing panel that covers 4,813 clinically-relevant genes. We next sought to streamline testing and designed a customized panel of 55 genes (Illumina). Variants were annotated and classified and the most likely disease-causing variants were validated by Sanger sequencing in the patient and available affected and unaffected family members. Results Out of 61 patient's sequenced, unexpected pyruvate kinase (PK) deficiency were found in 11 patients. PK enzyme activity assay was within normal limits in all these cases. Three patients with G6PD deficiency, 2 patients with glucose-6-phosphate isomerase deficiency and 1 case with hexokinase deficiency were found. Of 5 patients with stomatocytes on peripheral blood film, 3 had Mediterranean stomatocytosis/ macrothrombocytopenia (ABCG5/ABCG8) and 2 were found to have overhydrated hereditary stomatocytosis (RHAG). We also found 6 cases of HA to have a mutation in erythrocyte membrane protein-coding genes. Heterozygous nonsense mutation in the ANK1 gene was found in 2 cases and compound heterozygous missense mutations in ANK1 were seen in 1. Missense mutations were also found in SPTB gene in other 3 cases. Xerocytosis or dehydrated hereditary stomatocytosis (PEIZO1) was found in 3 patients. Seventeen cases for suspected CDA were studied and SEC23B mutations were present in 7. In one patient only heterozygous mutation was found. There could be the possibility of intronic mutation or long deletion/insertion in her. Notably, 6 patients with CDA showed mutations in PKLR (n=3), 1 each had mutations in MTRR, SPTB and PIEZO1 genes. We also found a mutation in GATA1 gene in a patient of CDA with thrombocytopenia. Conclusion(s) HA/CDA showed highly varied etiology. Our experience demonstrates the high diagnostic yield (>75%) of targeted resequencing for molecular diagnosis of unexplained anemias. It is cost effective and can expedite the diagnosis where conventional testing is not helpful. Timely detection of the etiology was helpful in genetic counseling. It not only offered therapeutic benefits to our patients but may help us in future antenatal diagnosis if required. Therapeutic implications include performing splenectomy which can ameliorate the anemia in selected subgroups of patients (HS, PK deficiency, CDA type II). Disclosures No relevant conflicts of interest to declare.