The Diverse Genomic Landscape of Diamond–Blackfan Anemia: Two Novel Variants and a Mini-Review

Diamond–Blackfan anemia (DBA) is a ribosomopathy characterized by bone marrow erythroid hypoplasia, which typically presents with severe anemia within the first months of life. DBA is typically attributed to a heterozygous mutation in a ribosomal protein (RP) gene along with a defect in the ribosomal RNA (rRNA) maturation or levels. Besides classic DBA, DBA-like disease has been described with variations in 16 genes (primarily in GATA1, followed by ADA2 alias CECR1, HEATR3, and TSR2). To date, more than a thousand variants have been reported in RP genes. Splice variants represent 6% of identifiable genetic defects in DBA, while their prevalence is 14.3% when focusing on pathogenic and likely pathogenic (P/LP) variants, thus highlighting the impact of such alterations in RP translation and, subsequently, in ribosome levels. We hereby present two cases with novel pathogenic splice variants in RPS17 and RPS26. Associations of DBA-related variants with specific phenotypic features and malignancies and the molecular consequences of pathogenic variations for each DBA-related gene are discussed. The determinants of the spontaneous remission, cancer development, variable expression of the same variants between families, and selectivity of RP defects towards the erythroid lineage remain to be elucidated.


Introduction
Diamond-Blackfan anemia (DBA), an inherited bone marrow failure (IBMF) syndrome, is a ribosomopathy identified in humans.With an incidence of seven cases per million live births, DBA is characterized by congenital erythroid hypoplasia (erythroblastopenia) with severely reduced erythroid colony-forming units (CFU-E) [1].It typically presents with severe anemia within the first 2-3 months after birth (95% of patients diagnosed are <2 years of age; 99% are <5 years of age) [2].To date, Next-Generation Sequencing (NGS) approaches, including gene panels and Whole Exome Sequencing (WES), are considered the method of choice for the identification of pathogenic and likely pathogenic (P/LP) variants located in ribosomal protein (RP) genes [3] (Table 1).In 22% of patients with DBA features, no P/LP variants are detected (or no genetic diagnosis is achieved), while some patients with P/LP variants in RP genes display syndromic characteristics or a preleukemic state with no signs of anemia [1][2][3].Erythrocyte adenosine deaminase (eADA) levels are usually elevated in DBA, in contrast to a DBA phenocopy where biallelic CECR1 variants result in ADA2 deficiency [3,4].According to a position paper from the European Hematology Association, "DBA-like" disorders present with congenital red cell aplasia and intact ribosomal function [3,5].Table 2 describes DBA-like phenotypes, which involve erythroblastopenia and normocytic hyporegenerative anemia with normal eADA levels and no defects in rRNA maturation [2].Of note, less than half of DBA patients present with cardiac defects as well as dysmorphic features, including craniofacial malformations and the pathognomonic triphalangeal thumb.DBA patients have a 4.8-fold higher relative risk of developing solid tumors (including osteogenic sarcoma in children), myelodysplastic syndrome (MDS), and acute myeloid leukemia compared to the general population, with a cumulative incidence of 20% by 46 years of age [5][6][7].The role of TP53 in the latter trait of DBA is under investigation.The pathogenesis of DBA involves defects in ribosomal RNA (rRNA) maturation that provoke nucleolar stress, leading to the stabilization of p53 and activation of its targets, resulting in erythroid-specific cell cycle arrest and apoptosis.Defective erythropoiesis in DBA may also occur due to reactive oxygen species production by toxic free heme generated by the GATA1/HSP70 and globin/heme imbalance [2,8].DBA is inherited as an autosomal dominant trait with variable penetrance that prompts genetic counseling.The treatment of DBA involves corticosteroids after the first year of life.In contrast, non-responders and patients who develop significant toxicity to treatment, as well as infants, require regular red blood cell (RBC) transfusions with prompt screening and therapy for iron overload.Hematopoietic stem cell transplantation (HSCT) from matched siblings or unrelated donors is an alternative to chronic transfusions and achieves 91% overall survival in young children with DBA [2,5].
Although the clinical features of most DBA patients may be typical (macrocytic anemia, reticulocytopenia, and normal bone marrow cellularity with an absence of erythroid progenitors), diagnostic challenges include overlapping and atypical manifestations of BMF with variable penetrance.Two patients with novel variants in RP genes are herein reported and discussed in the context of genetic and phenotypic heterogeneity of DBA.

Case Series
Both of the patients were referred to Pediatric Hematology-Oncology Units in Greece, and their clinical description adheres to the CARE (CAse REports) guidelines (https:// www.equator-network.org/reporting-guidelines/care/,accessed on 20 September 2023).
Case #1.A 3-month-old male was admitted for poor feeding and marked pallor.Prenatal history was unremarkable.The infant was born at 35 weeks of gestation with low birth weight (normal for gestational age) and no dysmorphic features.At birth, there were no signs of anemia [hemoglobin (Hb) 13.6 g/dL], respiratory stress, or feeding problems.He presented at three months with severe anemia (Hb 1.8 g/dL).The patient developed cardiogenic shock despite immediate blood transfusions and required intensive care treatment.The initial diagnostic evaluation showed a reticulocyte index of 3.4% and normal bilirubin levels, while a smear of peripheral blood disclosed the presence of all white blood cell lineages with normal counts and morphology, increased platelets (644,000/µL) with variable size, and decreased red blood cells (RBC 0.47 million cells/µL, MCV 119.1 fL, MCH 38.3 pg, RDW 18.5%, reticulocytes 765/µL).The bone marrow aspirate revealed a complete absence of the erythroid lineage with the presence of hemophagocytic islets.Since there were no signs of hemolysis or blood loss, and a negative screening for infectious agents, congenital anemia associated with BMF, such as DBA, was highly suspected.Targeted Sanger sequencing of RPS19, RPL11, and RPL5 genes was negative, and subsequent WES analysis revealed a de novo heterozygous pathogenic variant in the RPS26 gene (NM_001029.5:c.181+1del;splice donor).The infant was placed on regular transfusions.At the last follow-up, he was receiving transfusions every three months with a pre-transfusion hemoglobin cut-off of 8-9 g/dL.
Case #2.A 4-month-old female presented with worsening pallor for three consecutive weeks.Personal and family histories were non-contributory.Development was normal for her age, and no congenital anomalies were present.Complete blood count revealed severe anemia (Hb 5.7 g/dL, MCV 97.8 fL, MCH 32.6 pg, reticulocytes 7200/µL, white blood cells 10,070/µL, and platelets 725,000/µL), while further laboratory investigations were significant only for low B12 vitamin levels (126 pg/mL).RBC transfusion and B12 administration resulted in a temporary hemoglobin increase (8.2 g/dL).As anemia relapsed and reticulocytopenia did not improve, the patient underwent a bone marrow aspiration, which showed normal cellularity with diminished erythroid precursors.Based on the latter findings, DBA was highly suspected.NGS was performed for 160 genes using the Illumina MiSeq System and SureSelect (Agilent, Santa Clara, CA, USA) and revealed a de novo heterozygous variant (c.156-1G>A) in the RPS17 gene.This G>A substitution is a novel splice variant related to DBA.The infant received monthly RBC transfusions to maintain Hb levels > 8-9 g/dL until 12 months.After that age, prednisolone was introduced with a satisfying hemoglobin response without further transfusions.
Haploinsufficiency in RP genes results in defective RP biosynthesis and maturation, initiating ribosomal stress and apoptosis of erythroid progenitor cells.The two de novo and novel heterozygous pathogenic splice variants comprising RPS26:c.181+1del and RPS17:c.156-1G>A were conclusive in both cases.Splice site variants are commonly con-Children 2023, 10, 1812 5 of 11 sidered as P/LP and predicted to lead to a null effect.Further functional studies via either RNA or protein analysis are required to delineate the underlying mechanisms [15].Doherty et al., 2010 were able to show that RPS26 variants (including splice alterations) affect the function of proteins involved in rRNA processing in DBA patients.In particular, siRNA-mediated knockout of RPS26, encoding a component of the 40S ribosomal subunit, in HeLa cells led to decreased levels of 18S rRNA, impaired production of the small subunit and processing of the pre-rRNA, and accumulation of 43S, 26S, and 18S-E pre-rRNAs, thus indicating defects in cleavages at both ends of the 18S rRNA [16].RPS26 mutations have been identified as independent risk factors for short stature in DBA patients, which in turn reflects the cumulative steroid doses administered [17].DBA-related RPS26 mutations are not likely to display physical malformations; however, multiple physical abnormalities were recorded in a patient with a frameshift variant in RPL26.As shown by Gazda et al. (2012), the truncated RPS26 was unlikely to associate with the nascent ribosomal subunits, halting the synthesis of the large ribosomal subunits and resulting in low levels of the free 60S subunit, formation of half-mers in the polysomes, and a decrease in the amount of the precursors to large subunit rRNA [18].On the other hand, RPS17 lies on top of the small ribosomal subunit near one end of the eukaryotic initiation factor eIF-2 binding site and in close vicinity to three out of five RPs (RPS13, RPS16, and RPS19) that are involved in eIF-2 binding [19].

The DBA Genetic Landscape
To date, 1346 variations have been recorded in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/;accessed 18 October 2023), with P/LP variants accounting for 23% of records.According to ClinVar, the molecular consequences of variations in the DBA-related genes were missense mutations (48%), frameshift mutations (12%), splice variants (6%), and nonsense mutations (6%).SNVs are the most prevalent variation type (78.9%), followed by deletions (9.7%), insertions (5.9%), duplications (4.7%), and indels (0.9%).Variants typically reside in a single gene (99.4%), and the variation size is short (<50 bps; 98.9%).Among P/LP variants, frameshifts are common (31.1%), followed by missense (19.8%), non-coding RNA (ncRNA; 17.4%), nonsense (15.2%), splice site (14.3%), and untranslated region variations (UTR; 2.1%).Figure 1 illustrates all RPs in the literature with variations associated with DBA, and Figure 2 describes the molecular consequences of P/LP variations for each DBA-related RP gene.The results obtained from the ClinVar data investigation confirm the findings from previous DBA registry reports.RPS19, RPL5, RPS26, and RPL11 are indisputably the most frequently mutated RP genes in all relevant publications [3,20,21].EuroDBA reported that as many as 90% of the mutations occur in only six genes (RPS19, RPL5, RPS26, RPL11, RPL35A, and RPS24), which is in agreement with the findings of this survey (87%) [21].At this point, we should clarify that the ClinVar and OMIM (Online Mendelian Inheritance in Man; https://www.omim.org/,accessed on 29 September 2023) approach was chosen due to the scarcity of published DBA registry reports on genetic defects, even though EuroDBA and DBAR have a satisfactory number of registered patients (1191 and 750, respectively) [21].Obviously, OMIM does not provide a list of all the literature on the subject and ClinVar may not include all DBA-related variants ever diagnosed, and this is why we insist on growing DBA consortia and networks.

DBA-like Disorders
Although DBA is mainly homogenous in phenotypic expression, 6% of cases that phenocopy DBA and DBA-like disorders harbor variants in non-ribosomal genes (such as GATA1 and ADA2; Table 2) [3,23].OMIM recognizes P/LP variants in TSR2 and HEATR3 genes as causative for DBA14 with MFD and DBA21, respectively [12,13].TSR2 is involved in the processing and maturation of rRNA and binds to RPS26 (mutated in DBA10 correspondingly).Treacher Collins syndrome, which bears resemblance to the MFD accompanying DBA and displays rRNA reduction, is not characterized by increased risk for cancer, which could be partly linked to altered genetic functions, as with TSR2, which represses the transcription of NF-kappa B and seems to be involved in apoptosis [13,24].Besides TSR2's predominant role in producing adequate ribosome levels, it also seems necessary for erythroid lineage commitment through ribosome levels [25].A significant amount of GATA1 variations have been associated with DBA, indicating common pathways with RPs.
Haploinsufficiency in RPs results in an impaired translation of GATA1 mRNA, thus

DBA-like Disorders
Although DBA is mainly homogenous in phenotypic expression, 6% of cases that phenocopy DBA and DBA-like disorders harbor variants in non-ribosomal genes (such as GATA1 and ADA2; Table 2) [3,23].OMIM recognizes P/LP variants in TSR2 and HEATR3 genes as causative for DBA14 with MFD and DBA21, respectively [12,13].TSR2 is involved in the processing and maturation of rRNA and binds to RPS26 (mutated in DBA10 correspondingly).Treacher Collins syndrome, which bears resemblance to the MFD accompanying DBA and displays rRNA reduction, is not characterized by increased risk for cancer, which could be partly linked to altered genetic functions, as with TSR2, which represses the transcription of NF-kappa B and seems to be involved in apoptosis [13,24].Besides TSR2's predominant role in producing adequate ribosome levels, it also seems necessary for erythroid lineage commitment through ribosome levels [25].A significant amount of GATA1 variations have been associated with DBA, indicating common pathways with RPs.
Haploinsufficiency in RPs results in an impaired translation of GATA1 mRNA, thus mediating the erythroid defect in DBA.Cells bearing both RP and GATA1 variants exhibit reduced proliferation and delayed erythroid differentiation.RP haploinsufficiency and the subsequent decrease in GATA1 levels affect the balance of globin-heme and result in the accumulation of free cytoplasmic heme in erythroid progenitors, increasing the p53dependent apoptosis in these cells.Moreover, GATA1 has 5 UTR features that predict poor translation initiation rates, rendering its mRNA susceptible to ribosome deficiency [25,26].ADA2 is the only non-RP gene associated with DBA diagnosis with exome-wide significance, but it is unclear whether there are overlapping pathways [3].Patients with ADA2 variants present with a lack of erythroid precursors with maturation arrest in infancy, consistent with pure red cell aplasia, but they do not exhibit dysmorphic traits and, most importantly, they feature normal rRNA maturation [3,27,28].However, if DBA is suspected, screening for ADA2 variants should follow negative results for RP genes because HSCT seems to be effective in these patients [3].NOP58 is required for the biogenesis of the 40S ribosomal subunit, and its defects are associated with growth arrest due to defects in early pre-rRNA processing events necessary for ribosome assembly, while specific HEATR3 variants initially impair the nuclear import of RPL5 and eventually impair ribosome biogenesis [12,29].DAAM1 is implicated in cell polarity mainly through the Wnt signaling pathway and is required for myocardial maturation and sarcomere assembly [30].The EPO R150Q mutant displays only a mild reduction in affinity for its receptor.Still, its altered binding kinetics can lead to biased downstream signaling (reduced JAK2-mediated phosphorylation) and can thereby cause the ineffective stimulation of erythroid cell proliferation and differentiation.Unlike DBA, the latter entity can be effectively treated with EPO supplementation [3,31].In the same context, the IKZF1 R381C variant was reported to have DBA-like features, an immune-related gastrointestinal phenotype, and an alteration in hematopoietic gene expression networks, while mutations in the MPL proto-oncogene of the thrombopoietin (TPO) receptor have been linked to elevated TPO and a DBA-like phenotype [14,32].Loss-of-function variants in the MYSM1 gene, which is a regulator of transcription, present with transfusion-dependent refractory anemia in early childhood in addition to mild thrombocytopenia and low NK-and B-cell counts.Notably, MYSM1knockout mice manifest a BMF phenotype after oxidative DNA damage and increased p53 expression [33].Similarly to MYSM1, ZNF699 is involved in the regulation of transcription by RNA polymerase II by enabling DNA-binding transcription factors.Since 2021, it has been known to cause DEGCAGS syndrome, with anemia reported in 8 out of 14 diagnosed patients [34].Loss-of-function variants in NHEJ1, which preferentially mediates the repair of DNA double-stranded breaks, are associated with dysmorphic facies and immunodeficiency, and roughly half of the patients demonstrate anemia-either autoimmune or following bone marrow aplasia [3,35].Aside from sideroblastic anemia, variants in the PUS1 gene, which plays an essential role in tRNA function and in stabilizing the secondary and tertiary structure of many RNAs, and in the SLC25A38 gene, which is required during erythropoiesis and is essential for the biosynthesis of heme, were associated with a DBAlike phenotype.Notably, the knockout of both genes results in anemia [36,37].Ultimately, de novo TP53-activating variants are well documented in inherited BMF syndrome 5, exhibiting in some cases DBA-like features [38].Ultimately, the Table 2 data suggest the extension of investigations in non-RP genes in cases that resemble DBA.

The Correlation of Genetic Defects with Phenotype
The vast majority (96.8%) of DBA-associated P/LP variations correspond to ten RP genes, as illustrated in Figure 1.Although most DBA cases are sporadic, approximately 40-45% are familial, with the majority exhibiting autosomal dominant inheritance and incomplete penetrance [18].In terms of inheritance, DBA14 with MFD is X-linked recessive, and DBA21 is autosomal recessive, but both entities account for only nine cases in the literature, i.e., less than 1% of reported cases [12,13].Congenital hypoplastic normochromic and, usually, macrocytic anemia is routinely found in all DBA cases, while eADA is elevated in 8 out of 10 DBA-affected individuals [3,39].Based on the ClinVar data (Table 1) and OMIM phenotypic series (105650; 606129; 610629; 612527; 612528; 612561; 612562; 612563; 613308; 613309; 614900; 615550; 615909; 300946; 606164; 617408; 617409; 618310; 618312; 618313; and 620072) for DBA, we estimated the prevalence of specific traits among DBA patients.Growth disorders are expected in one-third of the patients (mainly presenting as a failure to thrive, followed by short stature).Still, it is unknown how much anemia and corticosteroids affect this outcome.Intriguingly, a DBA cohort study reported short stature in 9% of patients before treatment initiation.The same study noted that nearly half of patients are expected to present with one or more congenital malformations at diagnosis [3].Head, neck, and facial malformations are quite common among DBA patients.Mandibular deformities (primarily micrognathia followed by retrognathia) are expected in 17% of cases, clefts in 15-20%, and Cathie facies (short nose, broad nasal bridge, widely-spaced eyes, and thick upper lip) in approximately 10% of affected patients.Of interest, ear malformations seem to have been underestimated in DBA, with congenital aural atresia and microtia associated with 8.5% of the patients in OMIM.Triphalangeal thumbs are considered pathognomonic for DBA but occur only in roughly 15% of patients.Even though triphalangeal thumbs outnumber other skeletal malformations in DBA (accounting for 19% of the total), other disease entities should be ruled out [40].Isolated thumb malformations are highly prevalent among patients with RPL11 variants, while RPL5 variants are associated with syndromic features, such as craniofacial, thumb, and heart deformities [41].Patients with mutations in large ribosomal subunit protein (RPL) genes display significant correlations with the incidence of malformations, higher eADA levels, and more severe outcomes, compared to patients with mutations in small ribosomal subunit protein (RPS) genes [42].A significant portion of DBA patients suffer from congenital heart conditions (15-20%), predominantly from either ventricular or atrial septal defect.Lossof-function mutations in RPS24 have been associated with congenital heart disease, even though they display incomplete penetrance [43].Moreover, cardiac complications due to iron overload in transfusion-dependent children with DBA constitute a significant issue [44].Regarding laboratory investigations, anemia can be accompanied by elevated HbF and reticulopenia, while neutropenia (though not profound) is under-recognized, accounting for 15% of reported DBA cases.Nine out of ten eligible DBA patients respond to glucocorticoid treatment, one-quarter of patients become steroid-dependent (especially patients with variants in RPL5, RPL11, and RPS24 genes), and 11-25% of patients achieve remission spontaneously [3,45].Risk for malignancies in DBA might be lower compared with other inherited BMF syndromes but malignancies affect a considerable portion of non-transplanted patients (5%).The observed/expected ratios of 4.8 for any malignancy, 44.7 for colon carcinoma, 9.4 for lung cancer, 42.4 for osteogenic sarcoma, 352 for MDS, and 28.8 for acute myeloid leukemia are high and impose challenges in genetic counseling [46].DBA variants in patients with cancer/MDS refer mainly to RPS19, RPL35A, RPL11, RPL5, RPS17, and GATA1 genes (18%, 13%, 10%, 5%, 3%, and 3%, respectively; 49% unknown) [47].

Conclusions
The individualized monitoring of DBA patients is crucial and requires an integrated multidisciplinary approach.Frequent complete blood counts and periodic bone marrow biopsy or aspiration are fundamental in the context of the early diagnosis of new cytopenias or BMF.Steroid-dependent and transfusion-dependent patients should be closely monitored for adverse effects and growth, while cancer surveillance imposes follow-up visits every 4-6 months for all DBA patients [48].Genetic counseling is also essential and, hopefully, the implementation of techniques unveiling cryptic splice and non-coding variations in RP genes, along with copy number variation (CNV) assays and NGS (WGS with RNA-seq or WES plus Sanger), will establish a genetic diagnosis in >90% of DBA

Figure 2 .
Figure 2. Molecular consequence of P/LP variations in RP genes.

Figure 2 .
Figure 2. Molecular consequence of P/LP variations in RP genes.

Table 1 .
The genetic landscape of DBA in RP genes.

Table 2 .
The genetic landscape of DBA-like disorders.