Somatic HLA Mutations Expose the Role of Class I-Mediated Autoimmunity in Aplastic Anemia and its Clonal Complications.

Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by Human Leukocyte Antigen (HLA)-restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole exome sequencing (WES), we recently identified two aAA patients with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the Major Histocompatibility Complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping we screened 66 aAA patients for somatic HLA class I loss. We found somatic HLA loss in eleven patients (17%), with thirteen loss-of-function mutations in HLA-A*33:03, HLA-A*68:01, HLA-B*14:02 and HLA-B*40:02 alleles. Three patients had more than one mutation targeting the same HLA allele. Interestingly, HLA-B*14:02 and HLA-B*40:02 were significantly overrepresented in aAA patients, compared to ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA, and establishes a novel link between aAA patients' immunogenetics and clonal evolution.

Hmga2 collaborates with JAK2V617F in the development of myeloproliferative neoplasms.

High-mobility group AT-hook 2 (HMGA2) is crucial for the self-renewal of fetal hematopoietic stem cells (HSCs) but is downregulated in adult HSCs via repression by MIRlet-7 and the polycomb-recessive complex 2 (PRC2) including EZH2. The HMGA2 messenger RNA (mRNA) level is often elevated in patients with myelofibrosis that exhibits an advanced myeloproliferative neoplasm (MPN) subtype, and deletion of Ezh2 promotes the progression of severe myelofibrosis in JAK2V617F mice with upregulation of several oncogenes such as Hmga2. However, the direct role of HMGA2 in the pathogenesis of MPNs remains unknown. To clarify the impact of HMGA2 on MPNs carrying the driver mutation, we generated ΔHmga2/JAK2V617F mice overexpressing Hmga2 due to deletion of the 3' untranslated region. Compared with JAK2V617F mice, ΔHmga2/JAK2V617F mice exhibited more severe leukocytosis, anemia and splenomegaly, and shortened survival, whereas severity of myelofibrosis was comparable. ΔHmga2/JAK2V617F cells showed a greater repopulating ability that reproduced the severe MPN compared with JAK2V617F cells in serial bone marrow transplants, indicating that Hmga2 promotes MPN progression at the HSC level. Hmga2 also enhanced apoptosis of JAK2V617F erythroblasts that may worsen anemia. Relative to JAK2V617F hematopoietic stem and progenitor cells (HSPCs), over 30% of genes upregulated in ΔHmga2/JAK2V617F HSPCs overlapped with those derepressed by Ezh2 loss in JAK2V617F/Ezh2Δ/Δ HSPCs, suggesting that Hmga2 may facilitate upregulation of Ezh2 targets. Correspondingly, deletion of Hmga2 ameliorated anemia and splenomegaly in JAK2V617F/Ezh2Δ/wild-type mice, and MIRlet-7 suppression and PRC2 mutations correlated with the elevated HMGA2 mRNA levels in patients with MPNs, especially myelofibrosis. These findings suggest the crucial role of HMGA2 in MPN progression.

Clonal hematopoiesis in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited telomeropathy most frequently caused by mutations in a number of genes all thought to be involved in telomere maintenance. The main causes of mortality in DC are bone marrow failure as well as malignancies including leukemias and solid tumors. The clinical picture including the degree of bone marrow failure is highly variable and factors that contribute to this variability are poorly understood. Based on the recent finding of frequent clonal hematopoiesis in related bone marrow failure syndromes, we hypothesized that somatic mutations may also occur in DC and may contribute at least in part to the variability in blood production. To evaluate for the presence of clonal hematopoiesis in DC, we used a combination of X-inactivation, comparative whole exome sequencing (WES) and single nucleotide polymorphism array (SNP-A) analyses. We found that clonal hematopoiesis in DC is common, as suggested by skewed X-inactivation in 8 out of 9 female patients compared to 3 out of 10 controls, and by the finding of acquired copy neutral loss-of-heterozygosity on SNP-A analysis. In addition, 3 out of 6 independent DC patients were found to have acquired somatic changes in their bone marrow by WES, including a somatic reversion in DKC1, as well as missense mutations in other protein coding genes. Our results indicate that clonal hematopoiesis is a common feature of DC, and suggest that such somatic changes, though commonly expected to indicate malignancy, may lead to improved blood cell production or stem cell survival. Am. J. Hematol. 91:1227-1233, 2016. © 2016 Wiley Periodicals, Inc.

Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis.

MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed that donor BM harboring the Mdm2C305F mutation possessed decreased repopulation capacity compared to WT BM, suggesting a functional stem cell deficit. These results suggest that there is a fine tuned balance in the interaction of ribosomal proteins with the MDM2/p53 axis which is important in normal hematopoiesis.

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Disrupted lymphocyte homeostasis in hepatitis-associated acquired aplastic anemia is associated with short telomeres.

Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which immune-mediated bone marrow failure (BMF) develops following an acute episode of seronegative hepatitis. Dyskeratosis congenita (DC) is an inherited BMF syndrome characterized by the presence of short telomeres, mucocutaneous abnormalities, and cancer predisposition. While both conditions may cause BMF and hepatic impairment, therapeutic approaches are distinct, making it imperative to establish the correct diagnosis. In clinical practice, lymphocyte telomere lengths (TL) are used as a first-line screen to rule out inherited telomeropathies before initiating treatment for AA. To evaluate the reliability of TL in the HAA population, we performed a retrospective analysis of TL in 10 consecutively enrolled HAA patients compared to 19 patients with idiopathic AA (IAA). HAA patients had significantly shorter telomeres than IAA patients (P = 0.009), including four patients with TL at or below the 1st percentile for age-matched controls. HAA patients had no clinical features of DC and did not carry disease-causing mutations in known genes associated with inherited telomere disorders. Instead, short TLs were significantly correlated with severe lymphopenia and skewed lymphocyte subsets, features characteristic of HAA. Our results indicate the importance of caution in the interpretation of TL measurements in HAA, because, in this patient population, short telomeres have limited specificity.

Genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia in children and young adults.

Myelodysplastic syndrome (MDS) is a clonal blood disorder characterized by ineffective hematopoiesis, cytopenias, dysplasia and an increased risk of acute myeloid leukemia (AML). With the growing availability of clinical genetic testing, there is an increasing appreciation that a number of genetic predisposition syndromes may underlie apparent de novo presentations of MDS/AML, particularly in children and young adults. Recent findings of clonal hematopoiesis in acquired aplastic anemia add another facet to our understanding of the mechanisms of MDS/AML predisposition. As more predisposition syndromes are recognized, it is becoming increasingly important for hematologists and oncologists to have familiarity with the common as well as emerging syndromes, and to have a systematic approach to diagnosis and screening of at risk patient populations. Here, we provide a practical algorithm for approaching a patient with a suspected MDS/AML predisposition, and provide an in-depth review of the established and emerging familial MDS/AML syndromes caused by mutations in the ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72 genes. Finally, we discuss recent data on the role of somatic mutations in malignant transformation in acquired aplastic anemia, and review the practical aspects of MDS/AML management in patients and families with predisposition syndromes.

In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

Dysregulation of the Transforming Growth Factor ß Pathway in Induced Pluripotent Stem Cells Generated from Patients with Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome with clinical features of red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in ribosomal protein genes but the pathogenic mechanism is still unknown. We generated induced pluripotent stem cells from DBA patients carrying RPS19 or RPL5 mutations. Transcriptome analysis revealed the striking dysregulation of the transforming growth factor ß (TGFß) signaling pathway in DBA lines. Expression of TGFß target genes, such as TGFBI, BAMBI, COL3A1 and SERPINE1 was significantly increased in the DBA iPSCs. We quantified intermediates in canonical and non-canonical TGFß pathways and observed a significant increase in the levels of the non-canonical pathway mediator p-JNK in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. Surprisingly, nuclear levels of SMAD4, a mediator of canonical TGFß signaling, were decreased in DBA cells due to increased proteolytic turnover. We also observed the up-regulation of TGFß1R, TGFß2, CDKN1A and SERPINE1 mRNA, and the significant decrease of GATA1 mRNA in the primitive multilineage progenitors. In summary our observations identify for the first time a dysregulation of the TGFß pathway in the pathobiology of DBA.

Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human patients have so far not shown defects in pseudouridylation or ribosomal RNA processing. None of the mutant iPS cells presented here show decreased pseudouridine levels in rRNA or defective rRNA processing suggesting telomere maintenance defects account for most of the phenotype of X-linked DC. Finally gene expression analysis of the iPS cells shows that WNT signaling is significantly decreased in all mutant cells, raising the possibility that defective WNT signaling may contribute to disease pathogenesis.

mRNA deadenylation and telomere disease.

Dyskeratosis congenita (DC) is an inherited BM failure disorder that is associated with mutations in genes involved with telomere function and maintenance; however, the genetic cause of many instances of DC remains uncharacterized. In this issue of the JCI, Tummala and colleagues identify mutations in the gene encoding the poly(A)-specific ribonuclease (PARN) in individuals with a severe form of DC in three different families. PARN deficiency resulted in decreased expression of genes required for telomere maintenance and an aberrant DNA damage response, including increased levels of p53. Together, the results of this study support PARN as a DC-associated gene and suggest a potential link between p53 and telomere shortening.

Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia.

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies.

Genetic predisposition syndromes: when should they be considered in the work-up of MDS?

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by cytopenias, ineffective hematopoiesis, myelodysplasia, and an increased risk of acute myeloid leukemia (AML). While sporadic MDS is primarily a disease of the elderly, MDS in children and young and middle-aged adults is frequently associated with underlying genetic predisposition syndromes. In addition to the classic hereditary bone marrow failure syndromes (BMFS) such as Fanconi Anemia and Dyskeratosis Congenita, in recent years there has been an increased awareness of non-syndromic familial MDS/AML predisposition syndromes such as those caused by mutations in GATA2, RUNX1, CEBPA, and SRP72 genes. Here, we will discuss the importance of recognizing an underlying genetic predisposition syndrome a patient with MDS, will review clinical scenarios when genetic predisposition should be considered, and will provide a practical overview of the common BMFS and familial MDS/AML syndromes which may be encountered in adult patients with MDS.

Inherited bone marrow failure syndromes in adolescents and young adults.

The inherited bone marrow failure syndromes are a diverse group of genetic diseases associated with inadequate production of one or more blood cell lineages. Examples include Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia, thrombocytopenia absent radii syndrome, severe congenital neutropenia, and Shwachman-Diamond syndrome. The management of these disorders was once the exclusive domain of pediatric subspecialists, but increasingly physicians who care for adults are being called upon to diagnose or treat these conditions. Through a series of patient vignettes, we highlight the clinical manifestations of inherited bone marrow failure syndromes in adolescents and young adults. The diagnostic and therapeutic challenges posed by these diseases are discussed.

Baseline characteristics and disease burden in patients in the International Paroxysmal Nocturnal Hemoglobinuria Registry.

Paroxysmal nocturnal hemoglobinuria is a rare, acquired disease associated with hemolytic anemia, bone marrow failure, thrombosis, and, frequently, poor quality of life. The International PNH Registry is a worldwide, observational, non-interventional study collecting safety, effectiveness, and quality-of-life data from patients with a confirmed paroxysmal nocturnal hemoglobinuria diagnosis or detectable paroxysmal nocturnal hemoglobinuria clone, irrespective of treatment. In addition to evaluating the long-term safety and effectiveness of eculizumab in a global population, the registry aims to improve diagnosis, optimize patient management and outcomes, and enhance the understanding of the natural history of paroxysmal nocturnal hemoglobinuria. Here we report the characteristics of the first 1610 patients enrolled. Median disease duration was 4.6 years. Median granulocyte paroxysmal nocturnal hemoglobinuria clone size was 68.1% (range 0.01-100%). Overall, 16% of patients had a history of thrombotic events and 14% a history of impaired renal function. Therapies included anticoagulation (31%), immunosuppression (19%), and eculizumab (25%). Frequently reported symptoms included fatigue (80%), dyspnea (64%), hemoglobinuria (62%), abdominal pain (44%), and chest pain (33%). Patients suffered from poor quality of life; 23% of patients had been hospitalized due to paroxysmal nocturnal hemoglobinuria-related complications and 17% stated that paroxysmal nocturnal hemoglobinuria was the reason they were not working or were working less. This international registry will provide an ongoing, valuable resource to further the clinical understanding of paroxysmal nocturnal hemoglobinuria.