Analisi dell'Esoma mediante tecnologia Next Generation Sequencing in pazienti affetti da Eritrocitosi Idiopatica

Abstract Introduction: Erythrocytosis is a clinical condition characterized by increased hemoglobin (Hb) and/or hematocrit (Ht) >165 g/L and >49% for males and >160 g/L and >48% for females, respectively, that is associated with thrombohemorrhagic and cardiovascular complications. Causes may be primary such as polycythemia vera, a rare myeloproliferative neoplasm caused by the presence of a somatic mutation in the JAK2 gene of the erythroid progenitor cell, or secondary, caused in part by elevated erythropoietin (EPO) levels or a chronic hypoxic state. In most patients, the causes of secondary erythrocytosis are not yet known, and it is referred to as idiopathic erythrocytosis (EI). Recently, new DNA sequencing techniques, termed Next Generation Sequencing (NGS), allow the identification of new genetic mutations that may allow a better diagnostic approach to learn about the molecular mechanisms of EI. Purpose of the study The purpose of this study was to fine-tune NGS whole exome sequencing (WES) technology for the analysis of germline genetic variants in patients with erythrocytosis for whom the causative gene is unknown. Materials and Methods. DNA was extracted and purified from buffy coats obtained from 21 patients with idiopathic erythrocytosis. Libraries for NGS sequencing were prepared by DNA fragmentation, adaptor ligation, enrichment, and capture of nucleic acid fragments by complete exome-specific probes ( approximately 20,000 genes). NGS sequencing was performed using the Next Seq 550 platform ( 50X coverage with 99.98% ) and bioinformatics analysis using CLC Genomics software with specific pipelines and workflows for germline mutation identification. The identified mutations were then analyzed in silico using specific softwares to predict the pathogenicity of the identified genetic variants. Results Missense mutations in the HFE , VHL, SPTA1, PIEZO1 SLC40A1 genes were identified, and some of these germline variants were validated by Sanger sequencing. Conclusion The results obtained demonstrate that NGS WES sequencing techniques are a valid strategy for the analysis of unknown genetic variants present in patients with idiopathic erythrocytosis.

Abstract Introduzione: L’eritrocitosi è una condizione clinica caratterizzata da aumento dell’emoglobina (Hb) e/o dell’ematocrito (Ht) rispettivamente >165 g/L e >49% per i maschi e >160 g/L e >48% per le femmine che si associa a a complicanze trombo-emorragiche e cardiovascolari. Le cause possono essere primarie come la policitemia vera, una rara neoplasia mieloproliferativa causata dalla presenza di una mutazione somatica del gene JAK2 della cellula progenitrice eritroide, o secondarie, causate in parte da elevati livelli di eritropoietina (EPO) o da una stato di ipossia cronica. Nella maggior parte di pazienti le cause di eritrocitosi secondaria non sono ancora note e si parla di eritrocitosi idiopatica (EI). Recentemente, le nuove tecniche di sequenziamento del DNA, definite Next Generation Sequencing (NGS), consentono di identificare nuove mutazioni genetiche che possono consentire un migliore approccio diagnostico per conoscere i meccanismi molecolari di EI. Scopo dello studio Lo scopo di questo studio è stato di mettere a punto la tecnologia di sequenziamento NGS “whole exome sequencing” (WES) per l’analisi di varianti genetiche germinali in pazienti affetti da eritrocitosi per le quali il gene causativo non è noto. Materiali e metodi Il DNA è stato estratto e purificato da buffy coat ottenute da 21 pazienti affetti da eritrocitosi idiopati