Biallelic Mutations Research Articles

Generation of two iPSC lines (MHHi001-A-12 and MHHi001-A-13) carrying biallelic truncating mutations at the 3′-end of SRCAP using CRISPR/Cas9

Non-Floating Harbour Syndrome (FLHS) neurodevelopmental disorder (NDD) is a recently described disorder caused by mutations in certain regions of the SRCAP gene. We generated two iPSC lines that contain truncating mutation on both alleles at the 3′-end of SRCAP using CRISPR/Cas9 technology. Both cell lines are pluripotent, differentiate into the 3 germ layers and contain no genomic aberrations or off-target modifications. The cell lines form part of a human disease model to investigate the effects of truncating mutations in different regions of SRCAP.

Germline bi-allelic SH2B3/LNK alteration predisposes to a neonatal juvenile myelomonocytic leukemia-like disorder.

Juvenile myelomonocytic leukemia (JMML) is a rare, generally aggressive myeloproliferative neoplasm affecting young children. It is characterized by granulomonocytic expansion, with monocytosis infiltrating peripheral tissues. JMML is initiated by mutations upregulating RAS signaling. Approximately 10% of cases remain without an identified driver event. Exome sequencing of two unrelated cases of familial JMML of unknown genetics and analysis of the French JMML cohort identified 11 patients with variants in SH2B3, encoding LNK, a negative regulator of the JAK-STAT pathway. All variants were absent from healthy population databases, and the mutation spectrum was consistent with a loss of function of the LNK protein. A stoploss variant was shown to affect both protein synthesis and stability. The other variants were either truncating or missense, the latter affecting the SH2 domain that interacts with activated JAK. Of the 11 patients, eight from five families inherited pathogenic bi-allelic SH2B3 germline variants from their unaffected heterozygous parents. These children represent half of the cases with no identified causal mutation in the French cohort. They displayed typical clinical and hematologic features of JMML with neonatal onset and marked thrombocytopenia. They had a hypomethylated DNA profile with fetal characteristics and did not have additional genetic alterations. All patients showed partial or complete spontaneous clinical resolution. However, progression to thrombocythemia and immunity-related pathologies may be of concern later in life. Bi-allelic SH2B3 germline mutations thus define a new condition predisposing to a JMML-like disorder, suggesting that JAK pathway deregulation is capable of initiating JMML, and opening new therapeutic options.

BRCA-mutated gastric adenocarcinomas are associated with chromosomal instability and responsiveness to platinum-based chemotherapy.

Homologous recombination defect is an important biomarker of chemotherapy in certain tumor types, and the presence of pathogenic or likely pathogenic mutations involving BRCA1 or BRCA2 (p-BRCA) mutations is the most well-established marker for the homologous recombination defect. Gastric cancer, one of the most prevalent tumor types in Asia, also harbors p-BRCA mutations. To investigate the clinical significance of p-BRCA mutations, we analyzed 366 gastric cancer cases through next-generation sequencing. We determined the zygosity of p-BRCA mutations based on the calculated tumor purity through variant allelic fraction patterns and investigated whether the presence of p-BRCA mutations is associated with platinum-based chemotherapy and a certain molecular subtype. Biallelic p-BRCA mutation was associated with better response to platinum-based chemotherapy than heterozygous p-BRCA mutation or wild type BRCA genes. The biallelic p-BRCA mutations was observed only in the chromosomal instability subtype, while all p-BRCA mutations were heterozygous in microsatellite instability subtype. In conclusion, patients with gastric cancer harboring biallelic p-BRCA mutations were associated with a good initial response to platinum-based chemotherapy and those tumors were exclusively chromosomal instability subtype. Further investigation for potential association with homologous recombination defect is warranted.

Role of circulating biomarkers in spinal muscular atrophy: insights from a new treatment era.

Spinal muscular atrophy (SMA) is a lower motor neuron disease due to biallelic mutations in the SMN1 gene on chromosome 5. It is characterized by progressive muscle weakness of limbs, bulbar and respiratory muscles. The disease is usually classified in four different phenotypes (1-4) according to age at symptoms onset and maximal motor milestones achieved. Recently, three disease modifying treatments have received approval from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), while several other innovative drugs are under study. New therapies have been game changing, improving survival and life quality for SMA patients. However, they have also intensified the need for accurate biomarkers to monitor disease progression and treatment efficacy. While clinical and neurophysiological biomarkers are well established and helpful in describing disease progression, there is a great need to develop more robust and sensitive circulating biomarkers, such as proteins, nucleic acids, and other small molecules. Used alone or in combination with clinical biomarkers, they will play a critical role in enhancing patients' stratification for clinical trials and access to approved treatments, as well as in tracking response to therapy, paving the way to the development of individualized therapeutic approaches. In this comprehensive review, we describe the foremost circulating biomarkers of current significance, analyzing existing literature on non-treated and treated patients with a special focus on neurofilaments and circulating miRNA, aiming to identify and examine their role in the follow-up of patients treated with innovative treatments, including gene therapy.

EPCO-44. RHABDOID TUMOR PREDISPOSITION SYNDROME DUE TO A GERMLINE SMARCB1 MUTATION IN TWO INFANT SIBLINGS

Abstract INTRODUCTION Rhabdoid tumors (RTs) are highly aggressive malignancies caused by a loss–of–function mutation in the SMARCB1 tumor suppressor gene on chromosome 22 that makes up the SW1/SNF chromatin remodeling complex involved in gene transcription. In rhabdoid tumor predisposition syndrome (RTPS), a biallelic germline SMARCB1 mutation markedly increases the risk of affected individuals developing early, aggressive, and rapidly progressing RTs, most commonly in the CNS, referred to as atypical teratoid rhabdoid tumors (AT/RT), and the kidneys, but oftentimes presents with synchronous lesions. In this case series, we present a family affected by RTPS with a recurrence of AT/RT in two infant siblings with the same germline SMARCB1 mutation. CASE SERIES DESCRIPTION An 8-month-old female was brought to the emergency department for 3 months of vomiting, developmental regression, and CN VI palsy. Brain imaging revealed a posterior fossa mass, acquired obstructive hydrocephalus, and spinal metastasis. Four years later, her 3-month-old sister was also brought in for evaluation for 2 months of vomiting, irritability, increasing head circumference, and vertical gaze palsy. Her brain imaging similarly showed a posterior fossa mass and acquired obstructive hydrocephalus, but no metastasis. Both siblings underwent subtotal tumor resections and ventriculoperitoneal shunt placement followed by chemotherapy. While on active treatment, they experienced rapid disease progression and ultimately succumbed to their illness. Genetic testing on the family identified the same in-frame deletion in the SNF5 homology domain of SMARCB1 and loss of heterozygosity of chromosome 22 in the two sisters; their mother was found to be a carrier. CONCLUSION This case series describes a family affected by RTPS, demonstrating the risk of familial recurrence in SMARCB1 germline mutations and emphasizing the need for genetic testing and counseling for these families should they wish to have additional children.

Developmental and epileptic encephalopathy 82 (DEE82) with novel compound heterozygous mutations of GOT2 gene

PurposeDevelopmental and Epileptic Encephalopathies (DEEs) are rare neurological disorders characterized by early-onset medically resistant epileptic seizures, structural brain malformations, and severe developmental delays. These disorders can arise from mutations in genes involved in vital metabolic pathways, including those within the brain. Recent studies have implicated defects in the mitochondrial malate aspartate shuttle (MAS) as potential contributors to the clinical manifestation of infantile epileptic encephalopathy. Although rare, mutations in MDH1, MDH2, AGC1, or GOT2 genes have been reported in patients exhibiting neurological symptoms such as global developmental delay, epilepsy, and progressive microcephaly. MethodIn this study, we employed exome data analysis of a patient diagnosed with DEE, focusing on the screening of 1896 epilepsy-related genes listed in the HPO and ClinVar databases. Sanger sequencing was subsequently conducted to validate and assess the inheritance pattern of the identified variants within the family. The evolutionary conservation scores of the mutated residues were evaluated using the ConSurf Database. Furthermore, the impacts of the causative variations on protein stability were analyzed through I-Mutant and MuPro bioinformatic tools. Structural comparisons between wild-type and mutant proteins were performed using PyMOL, and the physicochemical effects of the mutations were assessed using Project Hope. ResultsExome data analysis unveiled the presence of novel compound heterozygous mutations in the GOT2 gene coding for mitochondrial glutamate aspartate transaminase. Sanger sequencing confirmed the paternal inheritance of the p.Asp257Asn mutation and the maternal inheritance of the p.Arg262Cys mutation. The affected individual exhibited plasma metabolic disturbances, including hyperhomocysteinemia, hyperlactatemia, and reduced levels of methionine and arginine. Detailed bioinformatic analysis indicated that the mutations were located within evolutionarily conserved domains of the enzyme, resulting in disruptions to protein stability and structure. ConclusionHerein, we describe a case with DEE82 (MIM: # 618721) with pathologic novel biallelic mutations in the GOT2 gene. Early genetic diagnosis of metabolic epilepsies is crucial for long-term neurodevelopmental improvements and seizure control as targeted treatments can be administered based on the affected metabolic pathways.

Retrospective analysis of confirmed cases of pulmonary veno-occlusive disease: a single tertiary referral center study

Abstract Background Pulmonary veno-occlusive disease (PVOD) is a rare cause of pulmonary hypertension that primarily results in intimal fibrosis of the pulmonary venules and veins, the venous lesions are often associated with changes in interlobular septa, lung parenchyma, and pulmonary arterioles and arteries. Purpose We sought to study the clinical characteristics of patients with confirmed PVOD at a single large referral center. Methods We retrospectively reviewed data from a total of 675 patients with pulmonary hypertension (identified by ICD codes or search of text) from 2017 and 2022 from a database obtained from electronic medical records at our institution. Patients with group 2 pulmonary hypertension were excluded. Only 29 cases were confirmed by histology or genetic testing. Results Out of the 29 patients confirmed with PVOD, 28 (96.5%) had a characteristic histology (lung biopsy (55.1%), explanted lung for transplant (27.5%) and autopsy (13.7%)) and 1 (3.4%) had a positive genetic test for EIF2AK4 biallelic mutation. The age at diagnosis was 53±16 years, 14 (48.2%) were men, 2 (6.8%) had systemic scleroderma, 2 (6.8%) had psoriasis, 2 (6.8%) were exposed to cytotoxic agents, 16 (55.1%) were smokers and 2 (6.8%) had a family history of pulmonary hypertension. All patients had computed tomography of the chest but only 12 (41.3%) had all 3 typical features found in PVOD (ground glass opacities, interlobular septal thickening, and lymphadenopathy). The most common feature was interlobular septal thickening in 26 (89.6%), followed by ground glass opacities present in 25 (86.2%) and lymphadenopathy in 12 (41.3%) of the patients. At diagnosis, mean pulmonary artery pressure was 45±15 mmHg and pulmonary vascular resistance was 9±6 WU. The median DLCO was 41 (34-58) %. On echocardiogram, 14 (48.2%) had severely enlarged right ventricle (RV), RV function was ≥moderately reduced in 17 (58.6%), RV systolic pressure was 63±20 mmHg and 6 (20.6%) had pericardial effusion. Out of 21 (72.4%) patients who received pulmonary vasodilators, 4 (13.7%) received single agent. 10 (34.4%) received 2 agents, 6 (20.6%) 3 agents, and 1 (3.4%) received 4 agents. 35.7% of these treated patients reported an improvement or initial worsening of symptoms with subsequent improvement afterwards. 18 (62%) patients with PVOD died, 12 (41.3%) had a lung transplant and 6 (20.6%) were still alive without the transplant until the day data was collected. Mean duration from diagnosis to death and lung transplantation was 446 ± 405 days and 274±479 days respectively. Conclusion PVOD is a rare and lethal cause of pulmonary hypertension. When suspected, a trial of medical therapy should be initiated carefully. The evaluation for lung transplant should be considered early given its high mortality.

Comprehensive molecular assessment of mismatch repair deficiency in Lynch associated ovarian cancers using next generation sequencing panel

ObjectivesAbnormalities in mismatch repair have been described in ovarian cancer, but few studies have examined the causes of mismatch repair deficiency (MMRd). To address this, we completed targeted mutational and...

Generation of four gene-edited human induced pluripotent stem cell lines with mutations in the ATM gene to model Ataxia-Telangiectasia

Ataxia-Telangiectasia (A-T) is an autosomal recessive multi-system disorder caused by mutations in the ataxia-telangiectasia mutated (ATM) gene, resulting, among other symptoms, in neurological dysfunction. ATM is known to be a master controller of signal transduction for DNA damage response, with additional functions that are poorly understood. CRISPR/Cas9 technology was used to introduce biallelic mutations at selected sites of the ATM gene in human induced pluripotent stem cells (hiPSCs). This panel of hiPSCs with nonsense and missense mutations in ATM can help understand the molecular basis of A-T.

A Novel Truncating Mutation in PAX1 Gene Causes Otofaciocervical Syndrome Without Immunodeficiency

Otofaciocervical syndrome (OTFCS) is a rare genetic disorder of both autosomal recessive and autosomal dominant patterns of inheritance. It is caused by biallelic or monoallelic mutations in PAX1 or EYA1 genes, respectively. Here, we report an OTFCS2 female patient of 1st consanguineous healthy parents. She manifested facial dysmorphism, hearing loss, intellectual disability (ID), and delayed language development (DLD) as the main clinical phenotype. The novel homozygous variant c.1212dup (p.Gly405Argfs*51) in the PAX1 gene was identified by whole exome sequencing (WES), and family segregation confirmed the heterozygous status of the mutation in the parents using the Sanger sequencing. The study recorded a novel PAX1 variant representing the sixth report of OTFCS2 worldwide and the first Egyptian study expanding the geographic area where the disorder was confined.

Loss of EFL1 Which Causes a Shwachman-Diamond Syndrome Reduces Cell Proliferation and Alters Transcriptional Profiling during Neutrophil Differentiation

Introduction. Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Patients with SDS are predisposed to develop myeloid malignancy. Almost all individuals with SDS harbor biallelic mutations in SBDS. The partner of SBDS is EFL1, a ribosomal protein with GTPase activity. More than a dozen patients have been found with SDS due to biallelic mutations in EFL1. EFL1 serves to release EIF6, facilitating the assembly of the 80S ribosome from 60S and 40S ribosome subunits. Our lab has generated both sbds and efl1 null zebrafish, which phenocopy much of the human syndrome. We recently reported that Sbds and Efl1 depletion leads to upregulation of tp53 and cdkn1a ( p21) and accumulation of Eif6 in SDS zebrafish models, which is hypothesized to cause bone marrow failure. However, the mechanism(s) of EFL1's loss-of-function leading to neutropenia is not known. Methods. We used CRISPR/Cas9 editing to generate mutations in exon 17 of Efl1 of 32Dcl3, an IL-3-dependent murine myeloid cell line that can be differentiated to mature neutrophils. We analyzed the effects of Efl1 mutations on cell proliferation, survival, and differentiation with G-CSF. Immunoblotting and qPCR-based gene expression analysis were performed to identify affected mechanistic pathways. Results. Two clones were isolated and were sequenced to verify biallelic deleterious mutations. A clone with compound heterozygous inframe deletion and frameshift mutations showed trace expression of EFL1 (EFL1-KD) and the other with compound heterozygous frameshift mutations showed no expression (EFL1-KO). They presented a myeloblast-like morphology indistinguishable from parental cells. They proliferated significantly less than parental cells, although there was no increased apoptosis. Both EFL1-KD and KO cells showed significantly increased protein expression of EIF6 and transcriptional expression of Trp53 and Cdkn1a. EFL1 was downregulated during differentiation of parental 32Dcl3 cells to neutrophils, whereas EIF6 levels did not change. Withdrawal and replenishment of IL-3 from culturing media did not alter EFL1 protein expression. When EFL1-KD and KO were cultured in differentiating media, they did not proliferate and underwent greater apoptosis, particularly after 3 days of differentiation compared to parental 32Dcl3. The surviving EFL1-KD and KO matured into neutrophils after 7 days. Gene profiling for neutrophil differentiation identified equivalent expression of key transcriptional factors ( Cebpa, Cebpe, and Spi1) of EFL1-KD and KO in the maintenance media, except for Gfi1 which was two-fold and three-fold high in EFL1-KD and KO compared to parental cells, respectively. G-CSF induced significantly higher expression of Cebpa and lower expression of Cebpe in EFL1-KO from those in parental cells, respectively. Based on the observation that somatic genetic rescue by EIF6 mutations occurs in human SDS patients and animal models, we transfected 32Dcl3 cells with siRNA against Eif6. In parental 32Dcl3 cells, reduction of EIF6 caused slow cell growth; however, transfection of siRNA to Eif6 partially rescued proliferation of EFL1-KD and KO in both maintenance and differentiating media. Conclusions. Our results suggested that imbalance of late maturing factors of ribosome associated with SDS may cause decreased proliferation in myeloid-committed cells, thereby leading to neutropenia in SDS. Newly established Efl1-deficient cells recapitulated key observation from SDS patients, with an increased proapoptotic profile in hematopoietic cells. Because EFL1 has only been recognized recently as a rare cause of SDS, we do not know if EFL1 deficiency serves as a leukemia predisposition syndrome. We speculate that the aberrant normal neutrophil differentiation may form the foundation for myeloid neoplasia in SDS.

Biallelic PI4KA mutations Disrupt B Cell Mitochondrial Metabolism and Cause Hypogammaglobulinemia

Introduction Biallelic mutations in the PI4KA gene cause PI4KA-related disorder, a novel condition with neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus), gastrointestinal (GI; inflammatory bowel disease [IBD] and multiple intestinal atresia [MIA]) and immunological manifestations. PI4KA encodes phosphatidylinositol (PI) 4-kinase alpha, which catalyzes the first step of phosphoinositide metabolism, phosphorylating PI to PI4P (phosphatidylinositol 4-phosphate). Phosphoinositides collectively have fundamental signalling roles in the plasma membrane and other organelles. We collected comprehensive clinical and laboratory data on 13 patients and performed a multiomic strategy, combining transcriptome, proteome, lipidome and metabolome analyses of three patients' EBV-transformed cell lines to unravel the burden of B cell deficiency in PI4KA-related disorder and define the role of PI4KA in B cells. Results Recurrent or severe infections (8/13) and GI (either gastroesophageal reflux disease, MIA or IBD; 6/13) manifestations were frequent. Thyroid disease (either hypo- or hyperthyroidism; 2/13), juvenile idiopathic arthritis (JIA; 1/13) and non-Hodgkin lymphoma (1/13) were reported. At last follow up 11 patients were alive. Ten had either hypogammaglobulinemia, decreased absolute numbers of B cells or decreased B cell subsets. B cell subsets showed increased transitional (4/7), decreased naive (2/7) and decreased switched memory (4/7) B cells. B cell proliferation to T independent mitogens, specifically IgM + CpG or pokeweed, was impaired in 2/2. T-cell (either CD3+, CD4+ or CD8+) lymphopenia was detected in 4/13. T-cell proliferation was maintained in 5/5. NK cells were decreased in 6/13. Ig replacement therapy (4/13) and immunosuppressive treatment (due to JIA [1/13] or IBD [2/13]) were given. Lipidome analysis showed that different glycerophospholipids profile (i.e., phosphatidylcholine [PC], phosphatidylethanolamines [PE] and phosphatidylserine [PS]) was altered. Metabolome analysis and pathway enrichment analysis confirmed impaired phosphatidylethanolamine biosynthesis and additionally showed altered PIP metabolism and mitochondrial dysfunction (i.e., beta-oxidation of short and long fatty acids). Although the total number of mitochondria was not affected, the mitochondrial activity was significantly reduced. Integration analyses of proteomics, metabolomic and lipidomic data reveals biochemical mechanisms underlying biallelic PI4KA mutation. In particular, dysregulation of metabolic enzymes and pathways involved in glutathione metabolism and tricarboxylic acid cycle was found (Fig. 1). These metabolic impairments ultimately impacted on oxidative phosphorylation, thus disrupting mitochondrial integrity. Gene set enrichment analysis of differentially expressed genes suggested a skewing towards naive B cell gene sets. Pathways enrichment analysis of transcriptome and proteome showed enrichment in B cell receptor (BCR) pathway and complex, PI3K/mTOR pathway and phospholipids metabolism. PI4P and its metabolite PI(4,5)P2 are determinants of PM identity, specifically controlling cytoskeletal dynamics. Deranged basal actin organization was reverted after anti-IgM stimulation. Hyperactivation of PI3K targets (pAKT473, p4EBP1 and pS6) was observed. Dysregulation of RABGAP1, RAB8B and LAMP3 mRNA prompted us to investigate autophagy. Indeed, LC3 was increased either in the presence or absence of chloroquine, showing that PI4KA mutations enhanced autophagosome formation and inhibited its degradation. Conclusion Overall, by altering the production of several key lipids, biallelic PI4KA mutations disrupt B cell metabolism causing mitochondrial dysfunction, mTOR hyperactivation, increased autophagy and deranged cytoskeleton organization. PI4KA-related disorder is a novel syndromic inborn error of immunity causing B cell deficiency and hypogammaglobulinemia. Figure 1. Integration of proteomics, metabolomics and lipidomics reveals biochemical mechanisms underlying biallelic PI4KA mutations.

Insight into the Natural History of Hereditary Thrombotic Thrombocytopenic Purpura: Short- and Long-Term Outcomes in a Longitudinally Followed Large Patient Cohort of the International Hereditary TTP Registry

Background: Hereditary thrombotic thrombocytopenic purpura (hTTP), also known as Upshaw-Schulman syndrome, is an ultra-rare, thrombotic microangiopathy resulting from bi-allelic ADAMTS13 mutations and severe congenital ADAMTS13 deficiency. Clinical presentation includes thrombocytopenia, hemolytic anemia and symptoms of organ ischemia (primarily of brain, heart, gastrointestinal tract and kidneys) during acute episodes and pregnancy complications, but less explicit symptoms (abdominal discomfort, headaches, neurocognitive problems or fatigue) responding to plasma therapy are also common. Standard of care consists of replenishing ADAMTS13 using plasma/plasma-derived blood products to treat acute episodes as well as prophylactically to prevent morbidity and mortality. Improvement of hTTP disease management requires better understanding of the treatment burden and possible shortcomings as well as short- and long-term clinical outcomes. Aims: Description of the natural history of hTTP in a prospectively followed large patient cohort with a focus on demographic and clinical characteristics, as well as on the occurrence of acute TTP episodes, comorbidities and mortality. Methods: Eligible for the current study were patients with a confirmed hTTP diagnosis (lasting ADAMTS13 activity <10%, bi-allelic ADAMTS13 mutations, and exclusion of acquired, autoimmune TTP) enrolled before December 31, 2021. In an ambidirectional approach, the registry assesses the patients' detailed medical history at enrollment (retrospective data) and then prospective clinical, molecular and observational data through questionnaires completed by treating physicians during annual visits or at occurrence of acute TTP episodes. Patients were followed by the International hTTP Registry until December 31, 2022 (end of current analysis), death, or loss to follow-up, whichever occurred first and results were reported using descriptive statistics. Results: At the end of December 2021, the International hTTP Registry had enrolled 182 confirmed hTTP patients from 49 sites in 20 countries, who were followed for a median of 3.6 (range, 0-18.5) years. The cohort included more female (n=98, 53.8%) than male (n= 84, 46.2%) patients, a gender disparity observed in adult patients because of increased diagnosis in women related to pregnancy complications. The patients were mostly White/Caucasian (n=111, 61.0%), followed by Asian (n=50, 27.5%), Black (n=2, 1.1%) and other ethnicities (n=19, 10.4%). Although 46.2% of the patients reported first symptoms of hTTP in the perinatal period (0-3 months), the median ages (ranges) at diagnosis and registry enrollment were 19.1 (0-69.9), and 30.4 (0.1-75) years, respectively. Prior to enrollment, 85.7% of patients had experienced at least one acute TTP episode with a mean (SD) of 4.3 (4.5) reported events per patient. The medical history was notable for transient ischemic attacks or strokes in 31% of hTTP patients, followed by arterial hypertension in 20%, chronic kidney disease in 12%, migraines/severe headaches in 8.2%, and seizures in 7.7%, respectively. During prospective follow-up 82 of 182 patients (45.1%) experienced 265 acute TTP episodes resulting in an overall event rate of 296.7 (95% confidence interval [CI]: 262.0-334.6) per 1000 person-years. Eight hTTP patients died during follow-up, five of them were on regular plasma prophylaxis. The causes of death were acute TTP episodes associated with cerebral (n=3; patient ages 33, 44 and 52 years) or cardiac (n=3; 39, 49, and 56 years) events, and unknown in two patients (75 and 79 years). Conclusions: The clinical burden due to recurrent acute TTP episodes and resulting neurological, cardiovascular and renal sequelae and other comorbidities is significant. Delayed diagnosis and insufficient treatment likely contributed to the observed morbidity and early mortality. Awareness, timely diagnosis and sufficient ADAMTS13 replacement therapy (for acute episodes and prophylactically) are crucial for improving short- and long-term clinical outcomes and disease burden in hTTP.

Loss of Metabolic Control Beyond the Promyelocyte Stage Resolves Myeloid Maturation Arrest in Reticular Dysgenesis

Reticular Dysgenesis (RD) is a particularly grave syndromic form of severe combined immunodeficiency. The hematopoietic phenotype manifests in defective myeloid and lymphoid development with profound neutropenia and lymphopenia. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), which catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. ADP is then phosphorylated to adenosine triphosphate (ATP) during the OXPHOS process. While there are abundant other sources of APD, AK2 constitutes the only mitochondrial enzyme that contributes to AMP “recycling” by phosphorylation. Accordingly, AK2-deficient myeloid cells have been shown to exhibit increased AMP levels 1. Beyond this observation, the cellular basis for the combined hematopoietic defect in RD remains elusive. Using a CRISPR/Cas9 AK2 biallelic knock-out model in human hematopoietic stem and progenitor cells (HSPCs), we have previously shown that AK2-/- HSPCs recapitulate RD myelopoiesis defects in vitro. AK2-/- HSPCs exhibit a severe proliferative defect that becomes apparent at the myelocyte (MC) and neutrophil (NP) stages, while the promyelocyte (PM) stage appears relatively spared, consistent with observations in RD patients. Our prior metabolomic analysis showed that AK2-/- MCs not only have increased levels of AMP but up of 20-fold increased levels of IMP derived by converting excess AMP to IMP conversion via AMP deaminases (AMPDs). In addition, we have shown that beginning at the MC stage, AK2 -/- cells exhibit an increase in NADH:NAD+ ratio, consistent with reductive stress and decreased aspartate levels. Our new data revealed that expressing the aspartate transporter GLAST and/or the water-forming NADH oxidase LbNOX in AK2-/- HSPCs partially rescues differentiation but not the proliferation defect. It raises the possibility that reductive stress and aspartate deficiency compromise protein synthesis necessary for maturation, but are separate from the proliferative defect related to high IMP and AMP levels. There were no metabolomic abnormalities noted at the PM stage. PMs have a higher proliferative index than MCs and NPs. This raises the question why the most metabolically active stage is phenotypically spared by AK2 deficiency. To better understand why the metabolic abnormalities associated with RD selectively affect differentiation beyond the promyelocyte stage, we investigated key regulators of cellular metabolism and proliferation, i.e., AMPK, ACC1 and S6, a target of mTOR signaling, at PM, MC and NP stages. We found activation of AMPK, ACC1 and S6 only at the PM stage, while AMPK, ACC1 and S6 protein expression at MC and NP stages was virtually undetectable. Interestingly, AK2-/- PMs exhibited notably lower ATP synthesis rates compared to controls, while ATP synthesis in AK2-/- MCs and NPs was significantly higher than in controls of corresponding stages, paralleling the accumulation of AMP and IMP noted earlier. These findings raise the possibility that tight metabolic control at the promyelocyte stage prevents AMP levels from rising by curtailing OXPHOS metabolism in AK2-/- PMs. In contrast, lack of metabolic control beyond the PM stage permits unopposed OXPHOS activity, thereby allowing AMP and IMP levels to rise to toxic levels that interfere with proliferation. In our ongoing work, we are testing whether pharmacologically redirecting metabolism from OXPHOS to glycolysis with UK5099, could rescue AK2-/- myelopoiesis defects. UK5099 is a potent inhibitor of the mitochondrial pyruvate carrier, which facilitates pyruvate transport across the mitochondrial inner membrane to fuel the TCA cycle. Additionally, we developed a hematopoietic-specific and inducible Cre-mediated Ak2 knock-out mouse model, which recapitulates the profound neutropenia and lymphopenia observed in RD patients. This mouse model will allow us to corroborate our in vitro observations and to investigate if the same mechanistic underpinnings also apply to the lymphoid lineage. Rissone A, Weinacht KG, la Marca G, et al. Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress. J Exp Med. Jul 27 2015;212(8):1185-202. doi:10.1084/jem.20141286

Congenital Dyserythropoietic Anemia Type II: An Update from the Congenital Dyseryhtropoietic Anemia Registry of North America (CDAR)

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare anemias characterized by ineffective erythropoiesis and hemolysis. The CDA registry in North America (CDAR; NCT02964494) was established to facilitate natural history studies. Participants who do not have a genetic diagnosis are offered family-trio whole genome sequencing (WGS) and elect to provide samples to CDAR biorepository to support collaborative mechanistic studies. CDA type II (CDA-II), the most common CDA type, is an autosomal recessive disease caused by biallelic SEC23Bvariants. CDA-II presents with hemolytic anemia with suboptimal reticulocytosis, iron overload, splenomegaly, and characteristic binucleation in 10-30% of the bone marrow (BM) erythroblasts. Although CDA-II is a recessive disorder, only one SEC23B variant is found in some patients. Here, we present the genetic and clinical data of CDA-II patients enrolled in CDAR so far. As of July 2023, 169 individuals (85 patients; 84 unaffected family members) enrolled in CDAR. Nine have a confirmed CDA-II diagnosis. Six patients have biallelic SEC23B pathogenic variants and 3 have only one variant identified. WGS for the 3 patients with a single variant revealed rare deep-intronic candidate variants. Since SEC23Bis ubiquitously expressed, we studied SEC23B protein levels in patient-derived lymphoblastoid cell lines generated for the CDAR biorepository. Patients with a single SEC23B variant exhibited an expression level comparable to those with biallelic mutations (Figure). The candidate deep intronic variants are currently being investigated for pathogenicity. Seven patients presented with hemolytic anemia during infancy, but the age of diagnosis was delayed by a mean of 12.5 yrs (0.1 to 55 y.o). Blood smears showed mild macrocytosis, poikilocytosis, and several spherocytes congruent with an osmotic gradient ektacytometry resembling mild hereditary spherocytosis. BM examination, when performed, exhibited erythroid hyperplasia, prominent dyserythropoiesis, and binucleation in ≥ 10% of erythroblasts. Anemia severity was variable: 6 patients were transfusion-dependent, 1 received intermittent transfusions, and 2 did not need blood transfusions. Two became transfusion-independent after splenectomy at ages 6 and 7 y.o, and two underwent successful matched-sibling BMT at age 10 and 26 y.o, after adequate chelation (liver iron concentration [LIC]<7 mg/g) Iron overload, disproportionate to transfusions, was present in 6 patients; 5 received iron chelation. All 5 patients with available MRI results had hepatic hemosiderosis (LIC range 3.4 to 21.8 mg/g) and evidence of myocardial hemosiderosis (T2* range 6.3 to 20 ms). Ferritin values were variable (range 58 to 1631.5 ng/ml) and discordant from LIC and T2* measurements. Notably, ferritin underestimated iron overload burden: 2 patients with normal ferritin (58 and 160 ng/mL) had hepatic hemosiderosis (LIC 6.1 and 3.4 mg/g respectively), and patients with severe hepatic and myocardial hemosiderosis (LIC 16.6-21.8 mg/g; T2* 6.3-7.8 ms) had modest ferritin values 1475-1631 ng/mL. Iron overload was present even in a non-transfusion-dependent patient and worsened in a patient post-splenectomy, suggesting a role for ineffective erythropoiesis. To further investigate this possibility, we measured erythroferrone (ERFE), GDF-15, and hepcidin levels in 2 patients so far. ERFE and GDF-15 levels were remarkably higher than controls, and hepcidin-to-ferritin ratios were significantly lower, suggesting that high ERFE, caused by ineffective erythropoiesis, inappropriately suppressed hepcidin for the body's iron levels. In summary, CDA-II diagnosis is often delayed despite early onset of anemia. Genetic testing may expedite the diagnosis. The severity of anemia is variable and there are several patients requiring transfusions. The risk of iron overload, including myocardial overload, is high and appears to be related to ineffective erythropoiesis. While frequency of transfusions may improve post-splenectomy, iron overload is not determined by blood transfusions alone and does not resolve post-splenectomy. Serum ferritin underestimated the burden of tissue iron in CDA-II, suggesting that MRI scans should be employed early. Future studies may identify a role for SEC23B deep intronic variants in CDA-II patients with heterozygous variants.

Investigation of RFC1 tandem nucleotide repeat locus in diverse neurodegenerative outcomes in an Indian cohort.

An intronic bi-allelic pentanucleotide repeat expansion mutation, (AAGGG)400-2000, at AAAAG repeat locus in RFC1 gene, is known as underlying genetic cause in cases with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) and late-onset sporadic ataxia. Biallelic positive cases carry a common recessive risk haplotype, "AAGA," spanning RFC1 gene. In this study, our aim is to find prevalence of bi-allelic (AAGGG)exp in Indian ataxia and other neurological disorders and investigate the complexity of RFC1 repeat locus and its potential association with neurodegenerative diseases in Indian population-based cohorts. We carried out repeat number and repeat type estimation using flanking PCR and repeat primed PCR (AAAAG/AAAGG/AAGGG) in four Indian disease cohorts and healthy controls. Haplotype assessment of suspected cases was done by genotyping and confirmed by Sanger sequencing. Blood samples and consent of all the cases and detailed clinical details of positive cases were collected in collaboration with A.I.I.M.S. Furthermore, comprehension of RFC1 repeat locus and risk haplotype analysis in Indian background was performed on the NGS data of Indian healthy controls by ExpansionHunter, ExpansionHunter Denovo, and PHASE analysis, respectively. Genetic screening of RFC1-TNR locus in 1998 uncharacterized cases (SCA12: 87; uncharacterized ataxia: 1818, CMT: 93) and 564 heterogenous controls showed that the frequency of subjects with bi-allelic (AAGGG)exp are 1.15%, < 0.05%, 2.15%, and 0% respectively. Two RFC1 positive sporadic late-onset ataxia cases, one bi-allelic (AAGGG)exp and another, (AAAGG)~700/(AAGGG)exp, had recessive risk haplotype and CANVAS symptoms. Long normal alleles, 15-27, are significantly rare in ataxia cohort. In IndiGen control population (IndiGen; N = 1029), long normal repeat range, 15-27, is significantly associated with A3G3 and some rare repeat motifs, AGAGG, AACGG, AAGAG, and AAGGC. Risk-associated "AAGA" haplotype of the original pathogenic expansion of A2G3 was found associated with the A3G3 representing alleles in background population. Apart from bi-allelic (AAGGG)exp, we report cases with a new pathogenic expansion of (AAAGG)exp/(AAGGG)exp in RFC1 and recessive risk haplotype. We found different repeat motifs at RFC1 TNR locus, like AAAAG, AAAGG, AAAGGG, AAAAGG, AAGAG, AACGG, AAGGC, AGAGG, and AAGGG, in Indian background population except ACAGG and (AAAGG)n/(AAGGG)n. Our findings will help in further understanding the role of long normal repeat size and different repeat motifs, specifically AAAGG, AAAGGG, and other rare repeat motifs, at the RFC1 locus.

Prime Editing of the α-Thalassemia Hb Constant Spring Mutation

Hemoglobin Constant Spring (HbCS) is the most common non-deletional α-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. Although the α-globin is expressed from two copies of homologous genes ( HBA2 and HBA1), the constant spring (CS) mutation CD142 (UAA &amp;gt; CAA) occurs only in HBA2 gene that leads to the instability of both mRNA and protein in erythroid cells. The newly developed gene editing technology prime editors (PEs) can generate all types of substitutions, providing therapeutic potential in different types of diseases. However, the efficiency of primer editor in primary hematopoietic stem cells is still a big challenge and lack of study in the HbCS mutation. We first created the endogenous HbCS mutation in human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells, providing as a model for prime editing optimization. The protein of α cs chain (a larger protein with extra 31 amino acids) was detected in the model cells with HBA2 bi-allelic CS mutation and normal HBA1, which exhibited the delay of expansion, higher apoptosis rate and altered erythroid maturation markers after induced differentiation. To correct the CS mutation for converting HbCS to HbA, we performed ex vivo delivery of mRNA encoding the prime editor together with a pegRNA and a nick guide RNA into the Hb CS HUDEP2 model cells and achieved the successful in-situ correction with an efficiency of 39.883±9.720% of CS alleles without enrichment, while no measurable editing in the same loci of HBA1 as evaluated by both Sanger sequencing and NGS. More importantly, the correction resulted in a significant decrease of α cs chain, thereby alleviated erythroid phenotype after induced maturation with reducing the apoptosis rate. After that, we collected the primary Hb H-CS patients derived CD34 + hematopoietic stem/progenitor cells (HSPCs) and confirmed that the defect on erythroid expansion, differentiation in Hb H-CS patients derived CD34 + HSPCs compared to the normal donor, especially demonstrated by lower enucleation rate and higher apoptosis. Moreover, the protein of α cs chain was detected in the erythroblast from Hb H-CS CD34+ HSPCs. We further successfully corrected the CS mutation in Hb H-CS patient derived CD34 + HSPCs with an efficiency of 23.68% by mRNA electroporation. At the same time, the editing efficiency was achieved with minimal generation of insertions and deletions and a low level of Cas9-dependent DNA off-target editing. Importantly, we observed an ~23% decrease of α cs chain protein and significant restoration of the enucleation defect from the differentiated erythroblast after PE correction in CD34 + HSPCs. In summary, these results suggests that the delivery of PE mRNA together with synthesized pegRNA and nick sgRNA can successfully achieve the in-situ correction in Hb HbCS mutation site in the model cells and the primary Hb H-CS patient derived CD34 + HSPCs. Our results provide proof of principle for prime editing for its therapeutic potential in directly and precisely correction of the Hb-CS mutation.

Treatment and Follow-up Data for Young Patients with a Gne Defect and Congenital Thrombocytopenia

Congenital thrombocytopenia can be associated not only with a reduced production, but also with an accelerated degradation of platelets. Sialic acid binds to platelet surface glycoproteins and protects platelets from degradation via the Ashwell-Morell receptor. The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. According to OMIM alterations in GNE are autosomal recessive associated with adult-onset progressive GNE myopathy (with or without thrombocytopenia) and autosomal dominant with sialuria. In recent years, biallelic GNE mutations located mainly in the N-acetylmannosamine (ManNAc) kinase domain of GNE have been described in children/young adults with otherwise unexplained thrombocytopenia. Recently, we identified compound heterozygous GNE variants in a young girl (Pt.1) with life-threatening intracranial bleeding and severe congenital thrombocytopenia (lowest platelet count 5 x 10 9/L), who needed weekly platelet transfusions. We showed decreased α2,3 sialic acid and increased terminal galactose and α2,6 sialic acid moieties in the girl´s platelets. She received hematopoietic stem cell transplantation (HSCT) at 2,3 years, which normalized platelet counts and function. Follow-up data showed normalized platelet function (20 months after HSCT) and counts (20, 21, and 23 months after HSCT). Following HSCT, the platelet lectin profile was markedly different from before transplantation with an increase in α2,3 sialic acid recognizing lectins WGA and MAL I compared with data before transplantation, indicating that sialic acid moieties have been normalized. Additionally, we identified a biallelic GNE variant in two siblings (Pt.2 and Pt.3) affected by congenital thrombocytopenia. Pt.2 is a 1,8-year-old boy with thrombocytopenia since birth (lowest platelet count 6 x10 9/L) who needed weekly platelet transfusions. His 17-year-old sister (Pt.3) presented with lifelong thrombocytopenia and suspected chronic immune thrombocytopenia; however, therapy with immunoglobulin and steroids did not elevate the platelets. The parents and two other siblings were clinically not affected. Flow cytometry performed for the affected siblings showed severely decreased thrombin-induced platelet CD62 and CD63 exposure hinting at an impaired α- and δ-granule secretion. NGS identified a homozygous GNE variant (NM_001128227.3:c.1727G&amp;gt;C, p.Gly576Ala) with damaging prediction to be present in both siblings. The parents and two healthy siblings are heterozygous carriers of this variant. The nucleotide change c.1726G&amp;gt;C leading to Gly576Arg has been described compound heterozygous in a patient with GNE myopathy. For the sister (Pt.3) lectin binding analysis showed decreased α-2,3 sialylation (MAA) and increased terminal galactose (RCA120) expression on platelets. This pattern is consistent with loss of sialic acid synthesis and indicative of rapid platelet clearance. She had received therapy with romiplostim since she was 12, resulting in stable platelet counts (&amp;gt; 50 x10 9/L). At the age of 1 year, Pt.2 also started with romiplostim therapy. During the 8-month treatment, platelet counts were mainly stable above 30 x 10 9/L. Only two transfusions were necessary when platelet counts dropped to 18 and 11 x 10 9/L. In conclusion, this research elucidates novel associations between GNE mutations, specifically within the ManNAc kinase domain, and congenital thrombocytopenia. These findings were derived from studying three patients carrying such mutations, underscoring this discovery's potential broad relevance. Importantly, this alteration in the GNE gene, traditionally associated with GNE myopathy, prompts us to anticipate and closely monitor the potential future development of this neuromuscular condition in these patients. Of particular interest is the effectiveness of romiplostim in ameliorating thrombocytopenia in these cases. Both siblings in our study exhibited stable, acceptable platelet counts in response to this therapy, suggesting that romiplostim might offer a promising therapeutic strategy for managing congenital thrombocytopenia linked to GNE mutations.

Integrative Genomic and Transcriptomic Analysis Reveals Targetable Vulnerabilities in Angioimmunoblastic T-Cell Lymphoma

Follicular helper T-cell lymphoma of the angioimmunoblastic type (AITL) is associated with dismal prognosis. We performed functional genomic approaches including whole-exome sequencing (WES; n=119), transcriptomic (n=78) and methylation (n=40) analysis. We identified recurrent mutations in known epigenetic drivers ( TET2, DNMT3A, IDH2 R172), and also identified novel ones (TET3, KMT2D). Somatic mutation of all three epigenetic drivers ( TET2, IDH2, and DNMT3A) was associated with poor prognosis (p&amp;lt;.001). Mutations in genes regulating T-cell receptor (TCR) signaling ( CD28 VAV1, FYN, PLCG1) or activation ( RHOA G17V), and regulators of the PI3K pathway (PIK(3)C members, PTEN,PHLPP-1/-2) were also found. Genome-wide DNA-methylation analysis integrated with mRNA expression profiling also revealed epigenetic alterations in genes regulating TCR-RHOA/B/C or PI3K-signaling. TET2 loss was noted in 85% AITLs and was significantly associated with RHOA G17V, CD28 and IDH2 R172mutations. AITLs lacking RHOA G17V tended to have mutations regulating the JAK-STAT pathway ( JAK2, JAK3, STAT1, STAT3, SOCS1). RNA-seq analysis identified fusion transcripts in genes regulating TCR activation (8%), revealed a restricted TCR repertoire in the majority of cases (a=87%, b=72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated association of B-cells in the tumor-milieu with better prognosis (p=.006), while dendritic cells were associated with worse prognosis (p=.001), which was further validated by immunohistochemistry using CD20, CD68, and CD163 antibodies. RNA-seq and corresponding WES analysis of 12 AITL patient-derived-xenografts (PDX) showed that bi-allelic TET2 mutations, DNMT3A mutations or sub-clonal mutations ( PLCG1 PHLPP2) werepropagated in sequential passages. Gene signatures related to T FH (follicular helper) and T CM (central memory) were also well-maintained in secondary passages in PDX models. Gene signatures of late PDX passages (3 rd-5 th) were enriched with genes related to proliferation and metabolic reprogramming, and in an independent cohort of AITLs, high expression of T3/T5 related signatures was associated with worse outcome (p=0.02/p=0.009). Low mRNA expression of PHLPP2 predicted poor prognosis (p=.03) and engineered PHLPP2 loss showed enhanced PI(3)K activation and FOXO1 inactivation in CD4+ T-cells in-vitro. Thus, we defined the genomic landscape for AITL, which is largely characterized by epigenetic alterations, TCR signaling and PI3K/AKT dysregulation, which may be amenable for therapeutic targeting.

Clinical Characteristics of Patients with TP53 Multihit MDS and AML. a Single-Center Experience

Introduction TP53 is a tumor suppressor protein encoded by the TP53 gene that plays a pivotal role in maintaining genomic stability in response to DNA damage, activates DNA repair programs and triggers cell-cycle arrest. Mutations in TP53 (TP53-mut) are present in approximately 5-10% of de novo myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), and confer an extremely poor prognosis, irrespectively of the treatment administered. The frequency of TP53 abnormalities increases up to 25-40% in therapy-related MDS and AML, up to 70%-80% in patients with complex karyotype and in patients with loss of chromosome 17/17p, 5/5q, or 7/7q. “Multihit” TP53-mut present with the loss of both wildtype TP53-alleles, either through point mutations or larger aberrations to chromosome 17, in complex monosomal karyotypes. Recent data strongly support the concept that TP53mut, particularly multi-hit TP53, results in similarly poor clinical outcomes, regardless of its classification as MDS or AML, arguing for a revised TP53 mutant myeloid entity that includes both MDS and AML. The aim of our analysis is to assess the impact of the multihit TP53-mut on clinical outcomes according to the type of treatment received. We will particularly focus on the presence of mono or biallelic mutation status of TP53 mutation. Methods We retrospectively evaluated 42 patients with TP53 mutated MDS or AML diagnosed from October 2016 to June 2023 in our department at the Catalan Institute of Oncology-L'Hospitalet in Spain. We analyze the prognosis of these two diseases, focusing on single-hit or multi-hit status of TP53 mutation and treatments received. Results Median age at diagnosis was 71 years (range, 44-87 years) with a male predominance (67%). Thirty-five (84%) patients were diagnosed of AML and 7 (16%) of MDS. Among AML patients, 17% had a prior diagnosis of dysplasia, 78% were diagnosed of AML with dysplasia-related changes, 13% with therapy-related neoplasms, and 9% were AML with maturation and/or AML with mutated NPM1. Regarding MDS patients, 57% had an excess of blast-2 (EB2) MDS, 43% MDS multilineage dysplasia (MLD) and MDS with excess of blast-1(EB1). Complex karyotype was present in 79% of the patients. The median variant allele frequency (VAF) of TP53mut was 36%. Multi-hit TP53 status was observed in 24 patients (57%). In this group of multi-hit TP53mut median age was 72 years (range,44-81 years) with a male predominance (67%), the median VAF of TP53mut was 40%. Twenty (83%) patients had MDS diagnosis and 4 (17%) had AML diagnosis; all of them presented complex karyotype. In the whole group the most concurrent mutations associated with TP53 were TET2 in 17%, ASXL1 in 14% and DNMT3A, NRAS y CREBBP in 12% of patients. Intensive chemotherapy (IC) was administrated as first-line treatment in 17% of patients, 45% received hypomethylating agents (HMA) monotherapy, 24% venetoclax-based combination with HMA, and 14% exclusively supportive treatment. 16 patients (38%) achieved a complete response (CR), 44%, 31% and 25% with HMA+venetoclax, IC and with HMA monotherapy, respectively. Eight patients (19%) achieved a partial response (PR), 88% with HMA and 12% with IC, 7 patients (17%) stable disease, 86% with HMA monotherapy and 14% with HMA+venetoclax and 2 patients (5%) were refractory to IC. In 2 patients (5%), disease was not evaluable. The treatment related mortality was 4.2%. Median overall survival (OS) was 12.5 months [IC 95 % (0.0, 23.54)] and median progression free survival (PFS) was 9, 2 months [IC 95% (3.2, 32.6)]. Median OS of patients with multihit TP53 was 9.8 months, vs 25.7 months in patients with single-hit TP53-mut (P=0.026) (Figure1). Median PFS of the patients with multi-hit and single-hit TP53 was 9.1 and 11.6 months (P=0.07), respectively. The impact of multi-hit TP53 on OS and PFS according to the treatment received (HMA monotherapy vs HMA + venetoclax vs IC) was not statistically significant, just like in the single-hit TP53 (Figure1). Conclusions The presence of multihit TP53 -mut is a prognostic variable for survival in our group of patients analyzed with TP53-Mut. Survival is poor, around one year, in this group of patients independently on the treatment strategy received; this fact would call into question the benefit of IC with the toxicity it entails, in this group of patients. Further development of new effective therapies for multihit TP53-mut AML and MDS is needed.