Screening of Mutations Related to Hereditary Anemias Using NGS-Targeted Sequencing Panel

Hereditary forms of anemia arise due to defects in erythrocyte differentiation from bone marrow precursor cells, or shortened survival of erythrocytes. Both cases result in the deficiency of mature cells in the bloodstream. Defects in the erythrocyte membrane proteins (e.g. hereditary spherocytosis and elliptocytosis/ pyropoikilocytosis) are common, and they manifest in association with morphological changes in the red blood cells. Other causes of hemolytic anemias include hemoglobinopathies (e.g. sickle cell diseases, thalassemias, etc) and enzymopathies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency), caused by defects in metabolic enzymes. Hereditary anemia can also be caused by erythroid lineage disorders such as congenital dyserythropoietic anemias (CDA), characterized by distinct morphologic abnormalities of marrow erythroblasts. Diseases that result in hereditary anemias are difficult to diagnose, due to the large clinical and genetic heterogeneity. The use of conventional techniques for the systematic screening of all potentially implicated genes would be impractical, costly and time-consuming. To address this issue, we have customized a rapid comprehensive next-generation sequencing-based assay that evaluates 58 genes with published disease-causing mutations for future clinical application. Of these genes, 27 were related with RBC cytoskeletal disorders and enzymopathies, 8 were involved with CDAs and 23 genes were related with hemochromatosis, sideroblastic anemia and porphyria. Our panel covers the complete coding region, splice site junctions, and some promoter regions. Exon capture probes were designed with the aid of the Sure Design program (Agilent Technologies, Inc., CA, USA). Targeted gene capture and library construction for next-generation sequencing (NGS) were performed using Haloplex kit (Agilent Technologies, Santa Clara, USA). Enriched samples were then sequenced on an Illumina HiSeq 2500 sequencer with 150 base pair, paired-end reads. Overall, the quality scores obtained were high, with an average of 91% of the bases analyzed with >Q30. This means that the probability that a given base is wrong is less than 0.1%. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using Sure Call software (Agilent Technologies, Inc., CA, USA). The validation included the benchmark sample NA12878 (Coriell Institute, NJ, USA) in triplicate. All variants were detected using Haloplex amplification followed by NGS were compared to the results available in the coding region sequencing databases of the NA12878 sample. The panel showed a sensitivity of 98.7% and a specificity of 99.9%. We also tested our panel in 43 patients with hereditary anemia searching for causative variants. Possible pathogenic variants were identified in 37 patients (86%) and explained the etiology of the diseases in 69.8% of the cases. Variants in the β-spectrin (SPTB gene) were the most frequent cause of hereditary spherocytosis (HS) in our sample, identified in 13 out of the 26 patients with previous diagnosis of HS (50%). Variants in the ankyrin (ANK1) were the second most frequent cause of HS, identified in 6 cases. Variants in SPTA1, SLC4A1, PKLR, CDAN1, G6PD, HFE and KIF23 genes were also identified. Most of the identified variants were novel. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular characterization of a disease. The identification of pathogenic variants is essential for genetic counselling, pre-implantation genetic diagnosis and even for future gene therapy. The panel we developed proved to be robust, with very low false positive and negative results and successfully determined the molecular causes of hereditary anemia in most cases.