Adult‐Onset β‐Thalassemia Major as Acquired Imprinting Disorder

Thalassemias, the most common inherited anemias, are a heterogenous group of genetic disorders resulting from a decreased synthesis of alpha or beta chains of hemoglobin. Beta-thalassemia results from mutations in the HBB gene on chromosome 11, leading to reduced (β+) or absent (β0) synthesis of beta-globin chains. Over 200 different HBB-causing mutations have been identified, resulting in complex genotype–phenotype correlations and wide clinical presentations classified in three main clinical diagnoses, ranging from asymptomatic carriers (beta-thalassemia minor) to Non-Transfusion-Dependent Thalassemia (NTDT—Thalassemia Intermedia) and Transfusion-Dependent Thalassemia (TDT—Thalassemia Major) [1]. Late-onset beta-thalassemia major is extremely rare, with only five cases identified so far [2-4]. In all these cases, the recessive HBB mutation became homozygous due to somatic paternal uniparental isodisomy of the 11p15 chromosome region (patUPiD11p15) in blood cells. Four cases of late-onset NTDT have also been identified, all harboring a somatic maternal 11p15 deletion that causes the hemizygous state of a beta-globin mutation in blood [5-7]. Interestingly, maternal deletions and patUPiD of chr11p15 are typical molecular defects of the growth- and cancer-associated disorder, Beckwith-Wiedemann syndrome spectrum (BWSp, [8]), which results from the dysregulation of imprinted genes controlling growth and proliferation, including IGF2, expressed on the paternal chromosome, H19 and CDKN1C, both expressed on the maternal chromosome [9]. Here, we present the case of a man who developed adult-onset beta-thalassemia major due to a paternal nonsense HBB mutation becoming homozygous in the bone marrow following a clonal somatic segmental pat UPiD. This case highlights the contribution of imprinted genes in the pathogenesis of hemoglobinopathies and defines adult-onset Thalassemias as acquired imprinting defects. The male patient, a 45 years old beta-thalassemia carrier, was admitted to our Centre after the occasional finding of anemia and hyperbilirubinemia. He reported several episodes of mild jaundice not associated with hyperchromic urine, nor gallstones, associated with a mean hemoglobin (Hb) concentration of 12 g/dL, a mean corpuscular volume (MCV) of 65 fL, and a mean corpuscular hemoglobin concentration (MCHC) of 22 pg/dL. A glucose-6-phosphate dehydrogenase (G6PD) deficiency screening yielded normal results. Serological tests for hepatitis C virus (HCV) and hepatitis B virus (HBV) were negative. At our first examination, pallor, latent jaundice and enlarged spleen were noted, with the following hematological parameter: Hb 9.2 g/dL, MCV 73.4 fL, MCH 23 pg, reticulocytes 138 000/ul, total bilirubin 8.1 mg/dL (indirect 7.63), Lactate Dehydrogenase (LDH) 451 U/L, HbA2: 3.4%, HbF 39.8%, iron 224 μg/dL, transferrin 191 mg/dL, ferritin 1417 ng/mL (sat 60%), consumed haptoglobin, normal transaminases, normal direct and indirect Coombs test. Iron accumulation was revealed by SQUID with a Liver iron concentration (LIC) of 14.7 7 mg/g w.w. (n.v. < 3) mgFe/g liver dry weight. Abdominal ultrasound showed splenomegaly and liver steatosis, and spine MRI showed diffuse hematopoietic hyperplasia, not associated with masses of extramedullary ineffective hematopoiesis. Bone marrow biopsy at 48 years showed hypercellular bone marrow and erythroid expansion. Iron chelation therapy was started at 49 years with subcutaneous deferoxamine 22 mg/Kg/day, with a relevant decrease of LIC to 7 mg/g w.w. V at 50 years. Because of progressive hemoglobin decrease (Hb 7.4 mg/dL), regular blood transfusions were begun at 51 years. No mass of ineffective hematopoiesis developed, and the spleen volume remained stable. Given the peculiar evolution of the clinical presentation, the presence of HBB mutations was evaluated by Sanger sequencing of genomic DNA extracted from peripheral blood leukocytes (PBLs). The molecular analysis revealed a homozygous CD39 variant of the HBB gene (NM_000518.4(HBB):c.118C>T(Q40*)). Sanger sequencing was also performed on DNA derived from the patient's buccal swab and urinary sediment, which, unlike PBLs DNA, demonstrated the mutation in heterozygosity with no allelic imbalance (Figure 1A). The father of the patient was not available for segregation analysis, and the mother is not a carrier of the HBB mutation. SNP-array analysis was conducted on DNA extracted from bone marrow. Consistent with the homozygosity of the HBB mutation in PBLs, a UPiD, extending from 11p15.2 to 11p15.5 (GRCh37, chr11:219089–14 922 363) and including the beta-globin and the BWS gene cluster, was detected (Figure 1B). The absence of the HBB mutation in the mother's genome supported the hypothesis of a paternal origin of the UPiD. To confirm this, we performed DNA methylation analyses of two imprinting control elements that are differentially methylated regions (DMRs) between the two parental alleles: H19/IGF2:IG-DMR, which is normally methylated only on the paternal allele, and KCNQ1OT1:TSS-DMR, which is methylated exclusively on the maternal allele. In the proband's PBL DNA, methylation profiling revealed a markedly increased methylation level at H19/IGF2:IG-DMR and a markedly reduced level at KCNQ1OT1:TSS-DMR, whereas three healthy controls showed the expected ~50% methylation for both DMRs (Figure 1C). These findings are consistent with a paternal UPiD of chromosome 11p15 (patUPiD11p15). The patient's bone marrow transcriptional profile was assessed by RNA sequencing. Two control RNA samples were obtained from the bone marrow of two individuals affected by hemoglobin H (HbH) and NTDT, respectively. The HbH case was a compound heterozygote for the alpha-globin variants: -3.7 (NG_000006.1:g.34164_37967del3804) and -CAL (NG_000006.1:g.8464_40664del32201). The NTDT case was a compound heterozygote for the beta-globin variants HBB:c.118C>T (CD39C>T) and HBB:c.-151C>T (−101 C>T), which only mildly affect beta-globin levels [10]. 2013 differentially expressed genes (DEGs), with 1441 upregulated and 572 downregulated, were identified by comparing the patient with the controls. The KEGG analysis showed that the most significantly upregulated genes were involved in the extracellular matrix (ECM)-receptor interaction pathway, while significantly downregulated genes were involved in the NF-κB signaling pathway, which leads to increased proliferation and accumulation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow [11]. To better understand the role of the patUPD11p15 in the etiology of the disease, we intersected the 2013 DEGs with the 308 genes located in the UPD region. Eighteen of these genes were differentially expressed, with 8 upregulated and 10 downregulated. Among the downregulated genes, we detected HBB and CDKN1C, while the upregulated genes included IGF2, the fetal hemoglobin (HbF) genes, HBG1 and HBG2, as well as their positive regulator, BGLT3 (Figure 1D). Finally, whole-exome sequencing (WES) was performed on DNA extracted from the patient's PBLs and urine sediment. The mutant and the normal HBB alleles were present at similar levels in the urine sediment, but a strong bias was present in the PBLs (mutant 92%, normal 8%). No further pathogenic variants or known hereditary persistence of fetal hemoglobin (HPFH)–associated variants were identified. Our study provides the first evidence that imprinted genes have a key role in the etiology of late-onset beta-thalassemia major. Similar to previous studies, we describe a man with a heterozygous β0 mutation paternally inherited, which has become homozygous in the bone marrow due to a somatic and tissue-specific patUPiD11p15. RNA-seq analysis performed in the patient's bone marrow demonstrated increased expression of the paternally expressed growth factor IGF2 and decreased expression of the maternally expressed CDKN1C, consistent with patUPiD11p15. This imbalance likely promoted clonal expansion of hematopoietic stem cells carrying the two copies of the paternal mutant allele, resulting in the transition from heterozygous carrier to homozygous and transfusion-dependent thalassemia (Figure 2). A case of BWS with congenital TDT has been previously reported [12]. This case also showed patUPiD11p15 and a paternal inherited nonsense CD39 HBB mutation in homozygosity, probably acquired in early embryogenesis. Three cases of late-onset sickle cell disease (SCD) have been associated with patUPiD11p15 originated within hematopoietic cells, suggesting similar molecular mechanisms with late-onset beta-thalassemia (Table 1 and refs. [13-15]). Thus, our study supports a role for chr11p15 imprinted genes in the pathogenesis of several late-onset hemoglobinopathies, including TDT, NTDT, and SCD. We propose classifying this group of blood disorders as acquired somatic tissue-specific imprinting disorders. Notably, maternal 11p15 UPiD has never been associated with these conditions, likely because such genetic changes leading to IGF2 downregulation and CDKN1C activation are detrimental to cell growth and thus do not promote clonal expansion. This insight has important clinical implications because it suggests that heterozygous carriers who inherit these mutations paternally have a higher risk of developing late-onset hemoglobinopathies. Thus, although late-onset hemoglobinopathies are somatic genetic conditions that do not follow traditional Mendelian inheritance, the parent-of-origin inheritance of the mutation, characteristic of imprinting disorders, plays a crucial role in disease predisposition. It remains unclear which factors may influence the age of onset of the transfusion-dependent status. Previous studies excluded a causal role of the percentage of mosaicism but reported that female sex was associated with a more rapid progression to disease ([15]; Table 1). Our patient developed the disease in his fourth decade despite having the highest reported level of UPiD mosaicism in blood. Interestingly, he displayed elevated HbF levels (39.8%) in peripheral blood, alongside increased expression of the HBG1 and HBG2 genes in the bone marrow (Figure 1D; Table 1), which may explain his unusually delayed requirement for transfusion therapy (at 51 years of age), despite several years of overt disease. His HbF levels were the highest among the reported cases of late-onset hemoglobinopathies (Table 1). Overexpression of BGLT3, a long non-coding RNA that promotes gamma-globin activation [16], may have contributed to the delayed clinical progression. PatUPiD11p15 is associated with late-onset TDT, while maternal 11p15 deletions are linked to late-onset NTDT. Maternal deletions remove the active CDKN1C allele, whereas patUPiD11p15 results in both loss of the active CDKN1C allele and duplication of the active IGF2 allele. Furthermore, both genes have been shown to influence hematopoiesis [17, 18]. However, although reduced CDKN1C and increased IGF2 dosage both may contribute to disease progression, loss of the active CDKN1C allele alone, as occurs in maternal deletions, appears sufficient to drive the pathological phenotype. In conclusion, late-onset beta-thalassemia and SCD can be viewed as acquired imprinting disorders, in which progression from a carrier state to overt disease involves molecular mechanisms that affect hematopoietic cells. Specifically, these mechanisms include altered expression of imprinted genes such as increased IGF2 and/or decreased CDKN1C. Clinically, this suggests that paternal inheritance of the beta-globin mutations in heterozygous carriers might predispose individuals to developing late-onset disease, highlighting the importance of considering parent-of-origin-dependent effects in disease risk assessment and management strategies. Emilia D'Angelo, Giorgia Mandrile, Nicolò Tesio, Francesco Cecere, Teresa Ceglie, Rosa Maria De Maria, Simona Mellone, Mara Giordano, and Neha Sanjay Kargutar contributed to the data collection and data analysis. Andrea Riccio and Giovanni Battista Ferrero supervised the work. Emilia D'Angelo, Giorgia Mandrile, Flavia Cerrato, Andrea Riccio, and Giovanni Battista Ferrero drafted the manuscript. All authors contributed to critical revisions and provided approval of the submitted and final versions of the manuscript. We are grateful to Antonella Roetto, Luca Sbaiz, and Marco Barberis who performed the first molecular diagnostic tests. This work was supported by grants from The Italian Ministry of University and Research (MUR) under the PRIN 2022 programme (Project ID: 2022B2N2BY, awarded to A.R. and G.B.F.) and Fondazione AIRC per la ricerca sul cancro ETS” (IG 2020 ID 24405 to A.R.). This case report was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. The patient provided informed consent for the publication of this report, including relevant clinical details. All efforts have been made to maintain patient anonymity, and no identifiable information has been disclosed. Patient consent was obtained. The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.