Incidence of Atrx Mutations in Myelodysplastic Syndromes (MDS)
Authors: Herbaux Charles , Duployez Nicolas , Badens Catherine , Poret Nicolas , Gardin Claude , Eclache Virginie , Daliphard Sylvie , Murati Anne , Cony-Makhoul Pascale , Cheze Stephane , Beve Blandine , Prebet Thomas , Hunault-Berger Mathilde , Renneville Aline , Figeac Martin , Stamatoullas Aspasia , Bastard Christian , Fenaux Pierre , Preudhomme Claude , Rose Christian 
Published: October 2019

Abstract
INTRODUCTION
Acquired α-thalassemia MDS (ATMDS) is an acquired syndrome characterized by a somatic point mutation or splicing defect in the ATRX gene in patients with myeloid disorders, primarily MDS. No large series have assessed its incidence in these pathologies. The ATRX protein is a chromatin-remodeling factor that may act as a transcriptional cofactor and plays an important role in the epigenetic regulation of gene expression (1). The unusual microcytosis in the context of MDS is due to a reduced synthesis of α-globin related to a dramatic down regulation of α-globin gene expression by inactivating mutation in the ATRX gene (2). We tried to better determine the frequency of ATRXmutations in a MDS registry and a clinical trial cohort.
METHODS
We first used next-generation sequencing (NGS) on samples from 80 patients included in GFM-AZA-EPO-2008-01 study, a phase II study of azacitidine combined to EPO in IPSS low and int-1 risk MDS patients resistant to EPO. All coding exons of ATRXwere sequenced using the following methodology: Library preparation was performed using the Ion Ampliseq™ Library kit 2.0-384 LV (Life Technologies®). Template-positive Ion Sphere Particles™ were prepared using the Ion OneTouch™ 200 Template Kit v2 (Life Technologies®). Sequencing was performed with the Ion PGM™ 200 Sequencing Kit and the Ion 318™ Chip on the Personal Genome Machine® (PGM™) platform (Life Technologies®). Data files were independently analysed using two softwares: Torrent Browser™ (Life Technologies®) and SeqNext™ (JSI® Medical Systems).
We then focused on the GFM registry, started in 2003, which includes 2980 MDS patients (at diagnosis regardless of the MDS type) and searched by direct Sanger method for mutations in the ATRXgene in MDS with unexplained microcytosis (MCV lower or equal to 80), ie without iron deficiency, inflammatory disease or history of inherited hemoglobinopathy.
RESULTS
NGS of ATRX gene in the 80 patients included in GFM-AZA-EPO-2008-01 study found no mutation. Of the 2980 patients in the GFM register, 139 had MCV below 80, unexplained in 58 of them. Out of these 58 patients, 14 had available material for ATRX gene sequencing by Sanger method. 2/14 patients were already known to have ATMDS, a RCMD and a CMML. In the remaining 12 patients, a mutation was found in 4 cases: a missense mutation in exon 9, a missense mutation in exon 4, a deletion in intron 9 with a frame shift and an addition in exon 1. Those mutations, to our knowledge, had not yet been described in ATMDS.
Therefore, of 2980 patients with MDS, we described 6 ATMDS (5 males, 1 female), ie an incidence of 6/2980 or 0.2%. However, 6/14 or 43% of patients with unexplained microcytosis had a mutation of ATRXand only 14/58 patients with unexplained microcytosis could be sequenced, suggesting an incidence closer to 6x58/14/2980 or 0.8%. There was no recurrent mutation. Of the 6 patients (median age was 76 years), 1 had RCUD, 1 CMML, 1 RAEB-2 and 3 RAEB-1. All karyotypes were different: favorable (n=2), intermediate (n=3) and poor (n=1) and IPSS was low (n=1), int-1 (n=3) and high (n=1). Median survival was 31.5 months. In the CMML patient, microcytosis was found 7 years before diagnosis. In the other 5 patients, microcytosis was discovered concomitantly with MDS. In 4 patients, microcytosis disappeared during the disease course. 2 patients died from AML progression, 2 from other causes and 2 were still alive.
CONCLUSION
Our data confirm the low frequency of ATRX mutations in MDS: 0% in an unselected clinical trial cohort of 80 lower risk MDS, 0.2 to 0.8% in a multicenter registry of 2980 MDS and 42% of MDS with unexplained microcytosis in this same registry. We also confirmed that these mutations can be found in all types of MDS. Larger studies are required to determine the prognostic value of these rare and non recurrent ATRXmutations. Law MJ. Cell. 2010.Steensma DP, Blood. 2005

Disclosures
No relevant conflicts of interest to declare.