Gut microbiota and its relationship with early vascular ageing in a Spanish population (MIVAS study).

BACKGROUND: Gut microbiota and its by-products are increasingly recognized as having a decisive role in cardiovascular diseases. The aim is to study the relationship between gut microbiota and early vascular ageing (EVA). METHODS: A cross-sectional study was developed in Salamanca (Spain) in which 180 subjects aged 45-74 years were recruited. EVA was defined by the presence of at least one of the following: carotid-femoral pulse wave velocity (cf-PWV), cardio-ankle vascular index (CAVI) or brachial-ankle pulse wave velocity (ba-PWV) above the 90th percentile of the reference population. All other cases were considered normal vascular ageing (NVA). MEASUREMENTS: cf-PWV was measured by SphygmoCor® System; CAVI and ba-PWV were determined by Vasera 2000® device. Gut microbiome composition in faecal samples was determined by 16S rRNA Illumina sequencing. RESULTS: Mean age was 64.4 ± 6.9 in EVA group and 60.4 ± 7.6 years in NVA (p < .01). Women in EVA group were 41% and 53% in NVA. There were no differences in the overall composition of gut microbiota between the two groups when evaluating Firmicutes/Bacteriodetes ratio, alfa diversity (Shannon Index) and beta diversity (Bray-Curtis). Bilophila, Faecalibacterium sp.UBA1819 and Phocea, are increased in EVA group. While Cedecea, Lactococcus, Pseudomonas, Succiniclasticum and Dielma exist in lower abundance. In logistic regression analysis, Bilophila (OR: 1.71, 95% CI: 1.12-2.6, p = .013) remained significant. CONCLUSIONS: In the studied Spanish population, early vascular ageing is positively associated with gut microbiota abundance of the genus Bilophila. No relationship was found between phyla abundance and measures of diversity.

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation.

Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in GNE, SLC35A1, GALE and B4GALT is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.

GALE variants associated with syndromic manifestations, macrothrombocytopenia, bleeding, and platelet dysfunction.

GALE gene encodes the uridine diphosphate [UDP]-galactose-4-epimerase, which catalyzes the bidirectional interconversion of UDP-glucose to UDP-galactose, and UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine. In that way, GALE balances, through reversible epimerization, the pool of four sugars that are essential during the biosynthesis of glycoproteins and glycolipids. GALE-related disorder presents an autosomal recessive inheritance pattern, and it is commonly associated with galactosemia. Peripheral galactosemia generally associates with non-generalized forms or even asymptomatic presentations, while classical galactosemia may be related to complications such as learning difficulties, developmental delay, cardiac failure, or dysmorphic features. Recently, GALE variants have been related to severe thrombocytopenia, pancytopenia, and in one patient, to myelodysplastic syndrome.

What is the context? GALE gene encodes for the UDP-Galactose 4-Epimerase, an enzyme involved in the Leloir pathway of galactose catabolism and protein glycosylation.Homozygous or compound heterozygous GALE variants associate with the disorder known as galactosemia type III.Three types of galactosemia can be distinguished: the peripheral, the intermediate, and the generalized form, which associate with different clinical symptoms and GALE genetic variants.Peripheral form is considered benign, while the intermediate and the generalized form is associated with severe and syndromic manifestations, including learning difficulties, delayed growth, sensorineural hearing loss, and early-onset cataracts, among others.What is new? In the last few years, GALE variants have been linked to hematological manifestations, such as anemia, febrile neutropenia, and severe thrombocytopenia.To date, the only GALE variants described in patients presenting hematological disorders are GALE p.Arg51Trp, p.Lys78ValfsX32, p.Val128Met, p.Thr150Met, p.Leu223Pro, and p.Gly237Asp.The thrombocytopenia observed in GALE patients is associated with reduced GPIbα and ß1 integrin glycosylation and externalization to the megakaryocyte and platelet surface, disrupting the actin cytoskeleton remodeling.What is the impact? GALE is an essential protein for the correct megakaryocyte and platelet glycosylation.

Platelet transcriptome analysis in patients with germline RUNX1 mutations.

BACKGROUND: Germline mutations in RUNX1 can cause a familial platelet disorder that may lead to acute myeloid leukemia, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet dysfunction, and a high risk of developing acute myeloid leukemia or myelodysplastic syndrome. Discerning the pathogenicity of novel RUNX1 variants is critical for patient management. OBJECTIVES: To extend the characterization of RUNX1 variants and evaluate their effects by transcriptome analysis. METHODS: Three unrelated patients with long-standing thrombocytopenia carrying heterozygous RUNX1 variants were included: P1, who is a subject with recent development of myelodysplastic syndrome, with c.802 C>T[p.Gln268∗] de novo; P2 with c.586A>G[p.Thr196Ala], a variant that segregates with thrombocytopenia and myeloid neoplasia in the family; and P3 with c.476A>G[p.Asn159Ser], which did not segregate with thrombocytopenia or neoplasia. Baseline platelet evaluations were performed. Ultrapure platelets were prepared for platelet transcriptome analysis. RESULTS: In P1 and P2, but not in P3, transcriptome analysis confirmed aberrant expression of genes recognized as RUNX1 targets. Data allowed grouping patients by distinct gene expression profiles, which were partitioned with clinical parameters. Functional studies and platelet mRNA expression identified alterations in the actin cytoskeleton, downregulation of GFI1B, defective GPVI downstream signaling, and reduction of alpha granule proteins, such as thrombospondin-1, as features likely implicated in thrombocytopenia and platelet dysfunction. CONCLUSION: Platelet phenotype, familial segregation, and platelet transcriptomics support the pathogenicity of RUNX1 variants p.Gln268∗ and p.Thr196Ala, but not p.Asn159Ser. This study is an additional proof of concept that platelet RNA analysis could be a tool to help classify pathogenic RUNX1 variants and identify novel RUNX1 targets.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY.

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

A Novel GATA1 Variant in the C-Terminal Zinc Finger Compared with the Platelet Phenotype of Patients with A Likely Pathogenic Variant in the N-Terminal Zinc Finger.

The GATA1 transcription factor is essential for normal erythropoiesis and megakaryocytic differentiation. Germline GATA1 pathogenic variants in the N-terminal zinc finger (N-ZF) are typically associated with X-linked thrombocytopenia, platelet dysfunction, and dyserythropoietic anemia. A few variants in the C-terminal ZF (C-ZF) domain are described with normal platelet count but altered platelet function as the main characteristic. Independently performed molecular genetic analysis identified a novel hemizygous variant (c.865C>T, p.H289Y) in the C-ZF region of GATA1 in a German patient and in a Spanish patient. We characterized the bleeding and platelet phenotype of these patients and compared these findings with the parameters of two German siblings carrying the likely pathogenic variant p.D218N in the GATA1 N-ZF domain. The main difference was profound thrombocytopenia in the brothers carrying the p.D218N variant compared to a normal platelet count in patients carrying the p.H289Y variant; only the Spanish patient occasionally developed mild thrombocytopenia. A functional platelet defect affecting αIIbß3 integrin activation and α-granule secretion was present in all patients. Additionally, mild anemia, anisocytosis, and poikilocytosis were observed in the patients with the C-ZF variant. Our data support the concept that GATA1 variants located in the different ZF regions can lead to clinically diverse manifestations.

TRAF3 alterations are frequent in del-3'IGH chronic lymphocytic leukemia patients and define a specific subgroup with adverse clinical features.

Interstitial 14q32 deletions involving IGH gene are infrequent events in chronic lymphocytic leukemia (CLL), affecting less than 5% of patients. To date, little is known about their clinical impact and molecular underpinnings, and its mutational landscape is currently unknown. In this work, a total of 871 CLLs were tested for the IGH break-apart probe, and 54 (6.2%) had a 300 kb deletion of 3'IGH (del-3'IGH CLLs), which contributed to a shorter time to first treatment (TFT). The mutational analysis by next-generation sequencing of 317 untreated CLLs (54 del-3'IGH and 263 as the control group) showed high mutational frequencies of NOTCH1 (30%), ATM (20%), genes involved in the RAS signaling pathway (BRAF, KRAS, NRAS, and MAP2K1) (15%), and TRAF3 (13%) within del-3'IGH CLLs. Notably, the incidence of TRAF3 mutations was significantly higher in del-3'IGH CLLs than in the control group (p < .001). Copy number analysis also revealed that TRAF3 loss was highly enriched in CLLs with 14q deletion (p < .001), indicating a complete biallelic inactivation of this gene through deletion and mutation. Interestingly, the presence of mutations in the aforementioned genes negatively refined the prognosis of del-3'IGH CLLs in terms of overall survival (NOTCH1, ATM, and RAS signaling pathway genes) and TFT (TRAF3). Furthermore, TRAF3 biallelic inactivation constituted an independent risk factor for TFT in the entire CLL cohort. Altogether, our work demonstrates the distinct genetic landscape of del-3'IGH CLL with multiple molecular pathways affected, characterized by a TRAF3 biallelic inactivation that contributes to a marked poor outcome in this subgroup of patients.

Molecular Dissection of Structural Variations Involved in Antithrombin Deficiency.

Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type.

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling.

BACKGROUND: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. OBJECTIVES: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. PATIENTS/METHODS: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH-BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole-exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. RESULTS: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin-4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP-6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up-regulation of TPM2 and TPM3 mRNA levels. CONCLUSIONS: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4-related thrombocytopenia.

Expanding the genetic spectrum of TUBB1-related thrombocytopenia.

ß1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 ß1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged ß1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that ß1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered ß1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.

Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model.

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbß3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbß3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

Gut microbiota composition and arterial stiffness measured by pulse wave velocity: case-control study protocol (MIVAS study).

INTRODUCTION: Intestinal microbiota is arising as a new element in the physiopathology of cardiovascular diseases. A healthy microbiota includes a balanced representation of bacteria with health promotion functions (symbiotes). The aim of this study is to analyse the relationship between intestinal microbiota composition and arterial stiffness. METHODS AND ANALYSIS: An observational case-control study will be developed. Cases will be defined by the presence of at least one of the following: carotid-femoral pulse wave velocity (cf-PWV), Cardio-Ankle Vascular Index (CAVI), brachial ankle pulse wave velocity (ba or ba-PWV) above the 90th percentile, for age and sex, of the reference population. Controls will be selected from the same population as cases. The study will be developed in Primary Healthcare Centres. We will select 500 subjects (250 cases and 250 controls), between 45 and 74 years of age. Cases will be selected from a database that combines data from EVA study (Spain) and Guimarães/Vizela study (Portugal). MEASUREMENTS: cf-PWV will be measured using the SphygmoCor system, CAVI, ba-PWV and Ankle-Brachial Index will be determined using VaSera device. Gut microbiome composition in faecal samples will be determined by 16S ribosomal RNA sequencing. Lifestyle will be assessed by food frequency questionnaire, adherence to the Mediterranean diet and IPAQ (International Physical Activity Questionnaire). Body composition will be evaluated by bioimpedance. ETHICS AND DISSEMINATION: The study has been approved by 'Committee of ethics of research with medicines of the health area of Salamanca' on 14 December 2018 (cod. 2018-11-136) and the 'Ethics committee for health of Guimaraes' (Portugal) on 15 October 2019 (ref: 67/2019). All study participants will sign an informed consent form agreeing to participate in the study, in compliance with the Declaration of Helsinki and the WHO standards for observational studies. The results of this study will allow a better description of gut microbiota in patients with arterial stiffness. TRIAL REGISTRATION DETAILS: ClinicalTrials.gov, identifier NCT03900338.

Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia.

BACKGROUND: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established. METHODS: We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response. RESULTS: Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition. CONCLUSIONS: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.