Germline ERG haploinsufficiency defines a new syndrome with cytopenia and hematological malignancy predisposition.

The genomics era has facilitated discovery of new genes predisposing to bone marrow failure (BMF) and hematological malignancy (HM). We report the discovery of ERG as a novel autosomal dominant BMF/HM predisposition gene. ERG is a highly constrained transcription factor critical for definitive hematopoiesis, stem cell function and platelet maintenance. ERG colocalizes with other transcription factors including RUNX1 and GATA2 on promoters/enhancers of genes orchestrating hematopoiesis. We identified a rare heterozygous ERG missense variant in 3 thrombocytopenic individuals from one family and 14 additional ERG variants in unrelated individuals with BMF/HM including 2 de novo cases and 3 truncating variants. Phenotypes associated with pathogenic germline ERG variants included cytopenias (thrombocytopenia, neutropenia, pancytopenia) and HMs (acute myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia) with onset before 40 years. Twenty ERG variants (19 missense, 1 truncating) including 3 missense population variants were functionally characterized. Thirteen potentially pathogenic ETS domain missense variants displayed loss-of-function characteristics disrupting transcriptional transactivation, DNA-binding and/or nuclear localization. Selected variants overexpressed in mouse fetal liver cells failed to drive myeloid differentiation and cytokine-independent growth in culture, and to promote acute erythroleukemia when transplanted into mice, concordant with these variants being loss-of-function. Four individuals displayed somatic genetic rescue by copy neutral loss of heterozygosity. Identification of predisposing germline ERG variants has clinical implications for patient/family diagnosis, counselling, surveillance, and treatment strategies including selection of bone marrow donors or cell/gene therapy.

Generation of iPSC lines and isogenic gene-corrected lines from two individuals with RPS19-mutated Diamond-Blackfan anemia syndrome.

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder that typically presents in infancy as hypoplastic anemia and developmental abnormalities in approximately 50% of cases. DBAS is caused by haploinsufficiency in one of 24 ribosomal protein genes, with RPS19 mutations accounting for 25% of cases. We generated iPSC lines from two patients with different heterozygous RPS19 mutations (c.191T > C and c.184C > T) and isogenic lines in which the mutations were corrected by Cas9-mediated homology directed repair.

Reduced toxicity conditioning for hematopoietic stem cell transplantation in children with Diamond-Blackfan anemia.

Not available.

Diagnosis, treatment, and surveillance of Diamond-Blackfan anaemia syndrome: international consensus statement.

Diamond-Blackfan anaemia (DBA), first described over 80 years ago, is a congenital disorder of erythropoiesis with a predilection for birth defects and cancer. Despite scientific advances, this chronic, debilitating, and life-limiting disorder continues to cause a substantial physical, psychological, and financial toll on patients and their families. The highly complex medical needs of affected patients require specialised expertise and multidisciplinary care. However, gaps remain in effectively bridging scientific discoveries to clinical practice and disseminating the latest knowledge and best practices to providers. Following the publication of the first international consensus in 2008, advances in our understanding of the genetics, natural history, and clinical management of DBA have strongly supported the need for new consensus recommendations. In 2014 in Freiburg, Germany, a panel of 53 experts including clinicians, diagnosticians, and researchers from 27 countries convened. With support from patient advocates, the panel met repeatedly over subsequent years, engaging in ongoing discussions. These meetings led to the development of new consensus recommendations in 2024, replacing the previous guidelines. To account for the diverse phenotypes including presentation without anaemia, the panel agreed to adopt the term DBA syndrome. We propose new simplified diagnostic criteria, describe the genetics of DBA syndrome and its phenocopies, and introduce major changes in therapeutic standards. These changes include lowering the prednisone maintenance dose to maximum 0·3 mg/kg per day, raising the pre-transfusion haemoglobin to 9-10 g/dL independent of age, recommending early aggressive chelation, broadening indications for haematopoietic stem-cell transplantation, and recommending systematic clinical surveillance including early colorectal cancer screening. In summary, the current practice guidelines standardise the diagnostics, treatment, and long-term surveillance of patients with DBA syndrome of all ages worldwide.

Modified Delphi Panel Consensus Recommendations for Management of Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is a rare hematologic condition for which there is no clear management algorithm. A panel of 11 adult and pediatric experts on aplastic anemia was assembled and, using the RAND/UCLA modified Delphi panel method, evaluated >600 varying patient care scenarios to develop clinical recommendations for the initial and subsequent management of patients of all ages with SAA. Here we present the panel's recommendations to rule out inherited bone marrow failure (IBMF) syndromes, on supportive care prior to and during first-line therapy, and on first-line (initial management) and second-line (subsequent management) therapy of acquired SAA, focusing on when transplant versus medical therapy is most appropriate. These recommendations represent the consensus of 11 experts informed by published literature and experience. They are intended only as general guidance for experienced clinicians who treat patients with SAA and are in no way intended to supersede individual physician and patient decision-making. Current and future research should validate this consensus using clinical data. Once validated, we hope these expert panel recommendations will improve outcomes for patients with SAA.

Insights into the Molecular Mechanisms of Genetic Predisposition to Hematopoietic Malignancies: The Importance of Gene-Environment Interactions.

SUMMARY: The recognition of host genetic factors underlying susceptibility to hematopoietic malignancies has increased greatly over the last decade. Historically, germline predisposition was thought to primarily affect the young. However, emerging data indicate that hematopoietic malignancies that develop in people of all ages across the human lifespan can derive from germline predisposing conditions and are not exclusively observed in younger individuals. The age at which hematopoietic malignancies manifest appears to correlate with distinct underlying biological pathways. Progression from having a deleterious germline variant to being diagnosed with overt malignancy involves complex, multistep gene-environment interactions with key external triggers, such as infection and inflammatory stimuli, driving clonal progression. Understanding the mechanisms by which predisposed clones transform under specific pressures may reveal strategies to better treat and even prevent hematopoietic malignancies from occurring.Recent unbiased genome-wide sequencing studies of children and adults with hematopoietic malignancies have revealed novel genes in which disease-causing variants are of germline origin. This paradigm shift is spearheaded by findings in myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) as well as acute lymphoblastic leukemia, but it also encompasses other cancer types. Although not without challenges, the field of genetic cancer predisposition is advancing quickly, and a better understanding of the genetic basis of hematopoietic malignancies risk affects therapeutic decisions as well as genetic counseling and testing of at-risk family members.

Clonal hematopoiesis in children with predisposing conditions.

Clonal hematopoiesis in children and young adults differs from that occuring in the older adult population. A variety of stressors drive this phenomenon, sometimes independent of age-related processes. For the purposes of this review, we adopt the term clonal hematopoiesis in predisposed individuals (CHIPI) to differentiate it from classical, age-related clonal hematopoiesis of indeterminate potential (CHIP). Stress-induced CHIPI selection can be extrinsic, such as following immunologic, infectious, pharmacologic, or genotoxic exposures, or intrinsic, involving germline predisposition from inherited bone marrow failure syndromes. In these conditions, clonal advantage relates to adaptations allowing improved cell fitness despite intrinsic defects affecting proliferation and differentiation. In certain contexts, CHIPI can improve competitive fitness by compensating for germline defects; however, the downstream effects of clonal expansion are often unpredictable - they may either counteract the underlying pathology or worsen disease outcomes. A more complete understanding of how CHIPI arises in young people can lead to the definition of preleukemic states and strategies to assess risk, surveillance, and prevention to leukemic transformation. Our review summarizes current research on stress-induced clonal dynamics in individuals with germline predisposition syndromes.

Spontaneous remission and loss of monosomy 7: a window of opportunity for young children with SAMD9L syndrome.

Monosomy 7 is the most common cytogenetic abnormality in pediatric myelodysplastic syndrome (MDS) and associated with a high risk of disease progression. However, in young children, spontaneous loss of monosomy 7 with concomitant hematologic recovery has been described, especially in the presence of germline mutations in SAMD9 and SAMD9L genes. Here, we report on our experience of close surveillance instead of upfront hematopoietic stem cell transplantation (HSCT) in seven patients diagnosed with SAMD9L syndrome and monosomy 7 at a median age of 0.6 years (range, 0.4-2.9). Within 14 months from diagnosis, three children experienced spontaneous hematological remission accompanied by a decrease in monosomy 7 clone size. Subclones with somatic SAMD9L mutations in cis were identified in five patients, three of whom attained hematological remission. Two patients acquired RUNX1 and EZH2 mutations during the observation period, of whom one progressed to myelodysplastic syndrome with excess of blasts (MDS-EB). Four patients underwent allogeneic HSCT at a median time of 26 months (range, 14-40) from diagnosis for MDSEB, necrotizing granulomatous lymphadenitis, persistent monosomy 7, and severe neutropenia. At last follow-up, six patients were alive, while one passed away due to transplant-related causes. These data confirm previous observations that monosomy 7 can be transient in young children with SAMD9L syndrome. However, they also indicate that delaying HSCT poses a substantial risk of severe infection and disease progression. Finally, surveillance of patients with SAMD9L syndrome and monosomy 7 is critical to define the evolving genetic landscape and to determine the appropriate timing of HSCT (clinicaltrials gov. Identifier: NCT00662090).

Biallelic inactivation of the NF1 tumour suppressor gene in juvenile myelomonocytic leukaemia: Genetic evidence of driver function and implications for diagnostic workup.

Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF-1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild-type allele. Here we examined the two-hit concept in leukaemic cells of 25 patients with JMML and NF-1. Ten patients with JMML/NF-1 exhibited a NF1 loss-of-function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound-heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF-1 and no variation in the JMML-associated driver genes PTPN11, KRAS, NRAS or CBL (JMML-5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound-heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF-1 reliably indicates a NF1-driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF-1.

Human hnRNPA1 reorganizes telomere-bound Replication Protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence Summary: At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

Germline landscape of RPA1, RPA2 and RPA3 variants in pediatric malignancies: identification of RPA1 as a novel cancer predisposition candidate gene.

Replication Protein A (RPA) is single-strand DNA binding protein that plays a key role in the replication and repair of DNA. RPA is a heterotrimer made of 3 subunits - RPA1, RPA2, and RPA3. Germline pathogenic variants affecting RPA1 were recently described in patients with Telomere Biology Disorders (TBD), also known as dyskeratosis congenita or short telomere syndrome. Premature telomere shortening is a hallmark of TBD and results in bone marrow failure and predisposition to hematologic malignancies. Building on the finding that somatic mutations in RPA subunit genes occur in ~1% of cancers, we hypothesized that germline RPA alterations might be enriched in human cancers. Because germline RPA1 mutations are linked to early onset TBD with predisposition to myelodysplastic syndromes, we interrogated pediatric cancer cohorts to define the prevalence and spectrum of rare/novel and putative damaging germline RPA1, RPA2, and RPA3 variants. In this study of 5,993 children with cancer, 75 (1.25%) harbored heterozygous rare (non-cancer population allele frequency (AF) < 0.1%) variants in the RPA heterotrimer genes, of which 51 cases (0.85%) had ultra-rare (AF < 0.005%) or novel variants. Compared with Genome Aggregation Database (gnomAD) non-cancer controls, there was significant enrichment of ultra-rare and novel RPA1, but not RPA2 or RPA3, germline variants in our cohort (adjusted p-value < 0.05). Taken together, these findings suggest that germline putative damaging variants affecting RPA1 are found in excess in children with cancer, warranting further investigation into the functional role of these variants in oncogenesis.

Germline predisposition traits in allogeneic hematopoietic stem-cell transplantation for myelodysplastic syndromes: a survey-based study and position paper on behalf of the Chronic Malignancies Working Party of the EBMT.

The recent application of whole exome or whole genome sequencing unveiled a plethora of germline variants predisposing to myeloid disorders, particularly myelodysplastic neoplasms. The presence of such variants in patients with myelodysplastic syndromes has important clinical repercussions for haematopoietic stem-cell transplantation, from donor selection and conditioning regimen to graft-versus-host disease prophylaxis and genetic counselling for relatives. No international guidelines exist to harmonise management approaches to this particular clinical scenario. Moreover, the application of germline testing, and how this informs clinical decisions, differs according to the expertise of individual clinical practices and according to different countries, health-care systems, and legislations. Leveraging the global span of the European Society for Blood and Marrow Transplantation (EBMT) network, we took a snapshot of the current European situation on these matters by disseminating an electronic survey to EBMT centres experienced in myelodysplastic syndromes transplantation. An international group of haematologists, transplantation physicians, paediatricians, nurses, and experts in molecular biology and constitutional genetics with experience in myelodysplastic syndromes contributed to this Position Paper. The panel met during multiple online meetings to discuss the results of the EBMT survey and to establish suggested harmonised guidelines for such clinical situations, which are presented here.

Hematopoietic cell transplantation and gene therapy for Diamond-Blackfan anemia: state of the art and science.

Diamond-Blackfan anemia (DBA) is one of the most common inherited causes of bone marrow failure in children. DBA typically presents with isolated erythroid hypoplasia and anemia in infants. Congenital anomalies are seen in 50% of the patients. Over time, many patients experience panhematopoietic defects resulting in immunodeficiency and multilineage hematopoietic cytopenias. Additionally, DBA is associated with increased risk of myelodysplastic syndrome, acute myeloid leukemia and solid organ cancers. As a prototypical ribosomopathy, DBA is caused by heterozygous loss-of-function mutations or deletions in over 20 ribosomal protein genes, with RPS19 being involved in 25% of patients. Corticosteroids are the only effective initial pharmacotherapy offered to transfusion-dependent patients aged 1 year or older. However, despite good initial response, only ~20-30% remain steroid-responsive while the majority of the remaining patients will require life-long red blood cell transfusions. Despite continuous chelation, iron overload and related toxicities pose a significant morbidity problem. Allogeneic hematopoietic cell transplantation (HCT) performed to completely replace the dysfunctional hematopoietic stem and progenitor cells is a curative option associated with potentially uncontrollable risks. Advances in HLA-typing, conditioning regimens, infection management, and graft-versus-host-disease prophylaxis have led to improved transplant outcomes in DBA patients, though survival is suboptimal for adolescents and adults with long transfusion-history and patients lacking well-matched donors. Additionally, many patients lack a suitable donor. To address this gap and to mitigate the risk of graft-versus-host disease, several groups are working towards developing autologous genetic therapies to provide another curative option for DBA patients across the whole age spectrum. In this review, we summarize the results of HCT studies and review advances and potential future directions in hematopoietic stem cell-based therapies for DBA.

Genomics of deletion 7 and 7q in myeloid neoplasm: from pathogenic culprits to potential synthetic lethal therapeutic targets.

Complete or partial deletions of chromosome 7 (-7/del7q) belong to the most frequent chromosomal abnormalities in myeloid neoplasm (MN) and are associated with a poor prognosis. The disease biology of -7/del7q and the genes responsible for the leukemogenic properties have not been completely elucidated. Chromosomal deletions may create clonal vulnerabilities due to haploinsufficient (HI) genes contained in the deleted regions. Therefore, HI genes are potential targets of synthetic lethal strategies. Through the most comprehensive multimodal analysis of more than 600 -7/del7q MN samples, we elucidated the disease biology and qualified a list of most consistently deleted and HI genes. Among them, 27 potentially synthetic lethal target genes were identified with the following properties: (i) unaffected genes by hemizygous/homozygous LOF mutations; (ii) prenatal lethality in knockout mice; and (iii) vulnerability of leukemia cells by CRISPR and shRNA knockout screens. In -7/del7q cells, we also identified 26 up or down-regulated genes mapping on other chromosomes as downstream pathways or compensation mechanisms. Our findings shed light on the pathogenesis of -7/del7q MNs, while 27 potential synthetic lethal target genes and 26 differential expressed genes allow for a therapeutic window of -7/del7q.

Massively parallel base editing to map variant effects in human hematopoiesis.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

European standard clinical practice - Key issues for the medical care of individuals with familial leukemia.

Although hematologic malignancies (HM) are no longer considered exclusively sporadic, additional awareness of familial cases has yet to be created. Individuals carrying a (likely) pathogenic germline variant (e.g., in ETV6, GATA2, SAMD9, SAMD9L, or RUNX1) are at an increased risk for developing HM. Given the clinical and psychological impact associated with the diagnosis of a genetic predisposition to HM, it is of utmost importance to provide high-quality, standardized patient care. To address these issues and harmonize care across Europe, the Familial Leukemia Subnetwork within the ERN PaedCan has been assigned to draft an European Standard Clinical Practice (ESCP) document reflecting current best practices for pediatric patients and (healthy) relatives with (suspected) familial leukemia. The group was supported by members of the German network for rare diseases MyPred, of the Host Genome Working Group of SIOPE, and of the COST action LEGEND. The ESCP on familial leukemia is proposed by an interdisciplinary team of experts including hematologists, oncologists, and human geneticists. It is intended to provide general recommendations in areas where disease-specific recommendations do not yet exist. Here, we describe key issues for the medical care of familial leukemia that shall pave the way for a future consensus guideline: (i) identification of individuals with or suggestive of familial leukemia, (ii) genetic analysis and variant interpretation, (iii) genetic counseling and patient education, and (iv) surveillance and (psychological) support. To address the question on how to proceed with individuals suggestive of or at risk of familial leukemia, we developed an algorithm covering four different, partially linked clinical scenarios, and additionally a decision tree to guide clinicians in their considerations regarding familial leukemia in minors with HM. Our recommendations cover, not only patients but also relatives that both should have access to adequate medical care. We illustrate the importance of natural history studies and the need for respective registries for future evidence-based recommendations that shall be updated as new evidence-based standards are established.