Skip to main content
  • Correspondence
  • Open access
  • Published:

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 SureSelectXT 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
figure 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, 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.

Availability of data and materials

The data supporting the findings of this study are available from the corresponding author upon request.



Alpha-thalassemia mental retardation X-linked


Acquired α-thalassemia myelodisplastic syndrome


α-globin gene


Novel single-nucleotide variant


Next-generation sequencing


Multiplex ligation-dependent probe amplification


American College of Medical Genetics and Genomics


  1. Steensma DP, Gibbons RJ, Higgs DR (2005) Acquired α-thalassemia in association with myelodysplastic syndrome and other hematologic malignancies. Blood 105:443–452.

    Article  CAS  PubMed  Google Scholar 

  2. Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S et al (2016) The GeneCards suite: from gene data mining to disease genome sequence analyses. CP Bioinform 54:1–30.

    Article  Google Scholar 

  3. Aguilera P, López-Contreras AJ (2023) ATRX, a guardian of chromatin. Trends Genet 39:505–519.

    Article  CAS  PubMed  Google Scholar 

  4. Ratnakumar K, Duarte LF, LeRoy G, Hasson D, Smeets D, Vardabasso C et al (2012) ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globin expression. Genes Dev 26:433–438.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gibbons RJ, Higgs DR (2000) Molecular-clinical spectrum of the ATR-X syndrome. Am J Med Genet 97:204–212.;2-X

    Article  CAS  PubMed  Google Scholar 

  6. Steensma DP, Higgs DR, Fisher CA, Gibbons RJ (2004) Acquired somatic ATRX mutations in myelodysplastic syndrome associated with α thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations. Blood 103:2019–2026.

    Article  CAS  PubMed  Google Scholar 

  7. Sessa R, Puzone S, Ammirabile M, Piscopo C, Pagano L, Colucci S et al (2010) Identification and molecular characterization of the-CAMPANIA deletion, a novel α0-thalassemic defect, in two unrelated Italian families. Am J Hematol 85:143–144.

    Article  PubMed  Google Scholar 

  8. Argentaro A, Yang J-C, Chapman L, Kowalczyk MS, Gibbons RJ, Higgs DR et al (2007) Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX. Proc Natl Acad Sci USA 104:11939–11944.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rehder C, Bean LJH, Bick D, Chao E, Chung W, Das S et al (2021) Next-generation sequencing for constitutional variants in the clinical laboratory, 2021 revision: a technical standard of the American College of Medical Genetics and genomics (ACMG). Genet Med 23:1399–1415.

    Article  PubMed  Google Scholar 

  10. Borges P, Pasqualim G, Matte U (2021) Which is the best in silico program for the missense variations in IDUA gene? A comparison of 33 programs plus a conservation score and evaluation of 586 missense variants. Front Mol Biosci 8:752797.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R et al (2019) VarSome: the human genomic variant search engine. Bioinformatics 35:1978–1980.

    Article  CAS  PubMed  Google Scholar 

  12. Garcia FADO, de Andrade ES, Palmero EI (2022) Insights on variant analysis in silico tools for pathogenicity prediction. Front Genet 13:1010327.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


Not applicable.


Not applicable.

Author information

Authors and Affiliations



Conceptualization was performed by MG; methodology by RC and RS; software by FR; validation by RC, RS, and ST; investigation by RC, RS, and ST; data curation by RC and FR; writing—original draft preparation by RC and FR; writing—review and editing by MG, MR, GP, TF, and SM; visualization by SM and SR; supervision by MG. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Michela Grosso.

Ethics declarations

Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of University of Naples Federico II (protocol code 443/21; date of approval: 24/02/2022).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Catapano, R., Russo, F., Rosetti, M. et al. Identification of a novel ATR-X mutation causative of acquired α-thalassemia in a myelofibrosis patient. Egypt J Med Hum Genet 25, 25 (2024).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: