5610480 TRANSFORMATION OF THALASSAEMIA GENETIC TESTING BY THE IMPLEMENTATION OF NGS-BASED THALASSEMIA ASSAY

Abstract

Background: The haemoglobinopathies, are the most common monogenic diseases worldwide, and have been recognized as a growing global health challenge. Thalassemia is a serious health problem in Cyprus where 12 % of the population are carriers of beta-thalassaemia and about 19% are carriers of alpha-thalassaemia[1]. Because of the high incidence of the disease and the heavy impact on the health care system, the Cyprus National Prevention Programme was initiated and is successfully running since 1978. The traditional strategy for thalassaemia molecular diagnosis was designed either to identify only the most common mutations, or to target the main causing genes of thalassaemia, HBA, and HBB by conventional molecular techniques such as PCR, Real-Time PCR, MLPA, Sanger sequencing, and Mini sequencing. The complex analysis with the use of multiple different techniques to obtain a result, can cause prolonged turn-around times and costly processes and may even not be conclusive. Aim: The aim is to replace the laborious conventional molecular testing with a comprehensive analysis of the thalassaemias by the establishment of an NGS-based test for the simultaneous genetic analysis of all variants on both HBA and HBB gene clusters. Methods: A commercial CE-IVD NGS Thalassemia kit, that detects all sequence variants in HBA1, HBA2, and HBB in a single run was evaluated. The assay detects SNVs, and Indels and also uses two independent methods in parallel to detect CNVs. For the initial evaluation, 25 DNA samples with known genotypes, previously characterised in our lab, were used. For the validation study, 100 DNA samples were analysed using the NGS Thalassemia kit in parallel with the conventional molecular methods. The results obtained were assessed and compared. Results: The initial evaluation of the NGS thalassemia assay demonstrated the strength of the assay to accurately detect all variations in the analysed genes. Moreover, the assay also detected variations that were not previously identifiable by our conventional multi-method strategy, Thus, we demonstrated the ability of the assay for complete analysis of thalassaemia. The assay was successfully validated: the results obtained by the NGS assay were in complete concordance with results from parallel analysis by our standard strategy. Hence, the NGS Thalassemia kit was successfully implemented in our daily routine practice. Summary/conclusion: The implementation of the NGS Thalassemia kit allows for comprehensive analysis for the haemoglobinopathies in a single run. It is very efficient compared to our traditional previously conducted thalassemia testing, yielding results of unprecedented quality, as well as saving time on three levels - for the physician, for the lab, and most importantly for the patient. Reference 1. Kountouris, P., et al, Scientific reports, 2016.6: p. 26371.