A new approach to Congenital Dyserythropoietic Anemias: toward a better definition of molecular mechanisms

Hereditary hemolytic anemias (HHAs) embrace a highly heterogeneous group of chronic disorders with a highly variable clinical picture. HHA encompass (1) hyporegenerative anemias (HAs), as congenital dyserythropoietic anemias (CDAs); (2) hemolytic anemias due to red cell membrane defects (HAMDs), as hereditary spherocytosis (HS) and hereditary stomatocytosis (HST). Although the workflow to diagnose these conditions is a normal clinical practice, differential diagnosis, classification, and patient stratification among HHAs are often very difficult. Beyond achieving a definitive diagnosis, knowing the genetic basis of these patients can be valuable also for guiding treatment. Next generation sequencing (NGS) refers to non-Sanger-based high-throughput DNA sequencing technologies. This technology plays a major role either in disease gene discovery or in clinical use for establishing a genetic diagnosis. Particularly, the major current application of NGS in diagnostics is through design of disease specific panel, named targeted (t)-NGS, in which a selected fraction of genes is sequenced. The primary aim of our study was the development of a fast, accurate, reliable and cost effective diagnostic/prognostic tool for HHAs based on t-NGS. In order to assess the reliability of this approach we created a t-NGS gene panel, named RedPlex, composed by 34 loci causative or candidates of HHAs. In silico design was performed by Agilent SureDesign web tool. For each locus, all coding regions, 5’ and 3’UTRs, and 100 bp flanking splice junctions were included. Sequence length was set at 150×2 nucleotides, and the predicted target size amounted to 538 regions (239.764 kb). Targeted enrichment was performed on 32 patients from 27 unrelated families by HaloPlex Target Enrichment System. High-throughput sequencing was performed by Illumina NextSeq 500. SureCall software was used for bioinformatic and computational analyses. RedPlex panel showed high sensitivity and specificity. It was able to capture at least 99.4% of 538 target regions with high and uniform coverage. We were able to obtain a conclusive diagnosis in approximately 72% of cases. In addition, a lot of patients (39%) showed multiple disease-associated variants suggesting complex inheritance. Indeed, t-NGS approach also allows the identification of “polygenic” genotypes, which may account for the phenotypic variability among HHA patients. Thus, the secondary aim of this project was the study of the interaction between mutated genes in HHA patients. We particularly focused on the functional interaction between two CDA-related genes, GATA1 and SEC23B. The demonstration of the direct interaction of GATA1 transcription factor on the SEC23B promoter provided also an explanation of the variability of phenotypes GATA1-related by means of the crosstalk of this gene with its target SEC23B