Identification of a novel ATR-X mutation causative of acquired α-thalassemia in a myelofibrosis patient

Dear Editor,

Acquired alpha-thalassemia mental retardation X-linked (ATRX) mutations are associated with the onset of α-thalassemia in several hematological malignancies including myelodysplasia, acute lymphoblastic leukemia, myelofibrosis, essential thrombocythemia, and acute myeloid leukemia (acquired α-thalassemia myelodisplastic syndrome, ATMDS) [1]. The ATRX gene (NM_000489.6) is located at Xq21.1 and encodes a chromatin remodeling protein which contributes to regulate the structure and function of chromatin in centromeric heterochromatin and telomeric domains to control different cellular pathways including DNA damage response and senescence mechanisms [2, 3]. ATRX is also involved in the epigenetic regulation of α-globin genes: loss-of-function mutations in the ATRX gene cause the transcriptional repression of the α-globin gene (HBA), thus resulting in a decreasing production of α-globin chains [4]. In this regard, mutations of the ATRX gene have been reported in association with a rare inherited pathology called X-linked α-thalassemia and mental retardation syndrome (or ATR-X syndrome) characterized by mental retardation, facial and urogenital abnormalities along with an α-thalassemia trait with elevated levels of β-globin or γ-globin tetramers (HbH or Barts' hemoglobin), the amount of which is directly related to the severity of the α-globin chain deficiency [5].

Here we report a novel single-nucleotide variant (SNV) in the ATRX gene, found by Next-Generation Sequencing (NGS) analysis in a 77-year-old Italian man previously healthy who had been hospitalized for myelofibrosis and was referred to our Centre to investigate the possible genetic cause of an acquired form of α-thalassemia with elevated levels of HbH. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Naples Federico II (project approval number 443/21). Genomic DNA was extracted using the Nucleon BACC3 kit (GE Healthcare, Life Sciences, Chicago, IL, USA) and analyzed by a customized NGS gene panel recently developed by our group to identify acquired or inherited mutations associated with thalassemic disorders. The DNA libraries were prepared with the SureSelect^{XT HS} Target Enrichment System kit (Agilent Technologies, Santa Clara, CA, USA) after enzymatic fragmentation and according to the manufacturer’s protocol. Library quality and quantity were checked with the TapeStation system (Agilent Technologies) and Qubit dsDNA High Sensitivity assay kit on Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Libraries were sequenced with MiSeq Reagent Kit v2 (300-cycles) by loading a concentrated pool (9 pM) and 1% Phix on a MiSeq Illumina® instrument (Illumina; San Diego, CA, USA). To exclude any kind of contamination, a blank negative control was included, and it followed all procedure’s steps, from DNA extraction to sequencing. Data analysis was performed using Alissa Report v1.1.6–2023-03 and Alissa Interpret v5.4.2 software (Agilent Technologies) and revealed the presence of a T > G transition at codon 520 in exon 7 of the ATRX gene (c.520T > G) with a variant allele frequency of 89.9% (179/199 variant coverage) which deviates from the expected values for germline mutations, thereby in agreement with the acquired origin of the variant. This SNV leads to a missense p.Cys174Gly mutation in the PHD-like domain, a hot-spot region for ATMDS defects [1, 6]. The mutation was confirmed by Sanger sequencing (Fig. 1A). NGS and MLPA analysis also excluded the presence of point mutations or large deletions in the α-globin gene cluster that are responsible of inherited α-thalassemia (Fig. 1B) [7].

Fig. 1

Analysis of the ATRX:c.520T > G variant. A Sanger sequencing with forward and reverse primers to confirm the presence of the novel mutation previously identified in the proband by NGS. The arrow indicates the mutated base; B MLPA analysis showing the absence of α-thalassemia deletions in the α-globin cluster, as previously described [7]; C Base conservation scores of 18 bases on the X chromosome’s negative strand of exon 7 of ATRX (and the respective amino acid encoded). Below each base, the PhyloP100way score from the VarSome database is presented in diagram form and color-coded. The PhyloP100way score calculation is based on multiple alignments of 99 sequences of genomes from different vertebrates compared to the human genome. It represents the conservation level of a specific nucleotide in the human genome: the higher the score, the more that nucleotide is conserved (red = highly conservated; yellow = moderately conservated; light green = mildly conservated; dark green = very mildly conservated). The asterisk indicates the position of the ATRX:c.520T > G variant (p.Cys174Gly) colored with red diagonal stripes

To our best knowledge and according to GnomAD exome, GnomAD genome, and ClinVar databases, this SNV is an unreported variant in the ATRX gene. Thirteen out of 18 in-silico prediction tools (CADD, Polyphen2 HVAR, Polyphen2 HDIV, FATHMM, M-CAP, MutPred, MVP, FATHMM-MKL, LRT, PrimateAI, PROVEAN, SIFT, SIFT4G) supported the possible pathogenicity of this SNV, whereas other five tools (BLOSUM, DANN, DEOGEN2, LIST-S2, MutationTaster) classified it as of uncertain significance (Table 1). In addition, six different meta-scores for in-silico pathogenicity assessment determined a very strong, strong, or moderate pathogenic prediction, basing on multiple tools as reported in Table 1. Furthermore, an analysis of base conservation scores on 99 vertebrate genome sequences aligned to the human genome (represented by PhyloP100way scores provided by the VarSome platform, https://varsome.com/about/resources/acmg-implementation) revealed that c.520T is a highly conserved nucleotide in the human genome, as represented in Fig. 1C. Indeed, this mutation falls in the PHD-like region of the protein, a functional domain where several other ATMDS mutations have been identified so far [8]. Based on this information, we classified this mutation as potentially pathogenic. In fact, according to the criteria of the American College of Medical Genetics and Genomics (ACMG), the detected SNV met three criteria which allow to establish its pathogenicity [9]: first, there are several computational systems supporting a possible deleterious effect of this mutation (PP1 rule); secondly, this mutation is located in a mutational hot-spot genomic area (PM1 rule); finally, no frequency data for this sequence variation are reported in the main genetic databases, such as the Exome Sequencing Project, 1000 Genome Project, or the Exome Aggregation Consortium (PM2 rule).

Table 1 Pathogenicity prediction meta-score

In conclusion, here we report a novel ATRX mutation in a patient with myelofibrosis in which the onset of HbH disease can be explained by impaired ATRX functions leading to altered expression of the α-globin genes. This report contributes to better define the ATRX gene mutational spectrum, with the purpose of improving genetic screening and diagnosis of rare diseases.