ABSTRACT
BACKGROUND: Autosomal dominant hypercholesterolemia (ADH) is due to deleterious variants in LDLR, APOB, or PCSK9 genes. Double heterozygote for these genes induces a more severe phenotype. More recently, a new causative variant of heterozygous ADH was identified in APOE. Here we study the phenotype of 21 adult patients, double heterozygotes for rare LDLR and rare APOE variants (LDLR+APOE) in a national wide French cohort. METHODS: LDLR, APOB, PCSK9, and APOE genes were sequenced in 5743 probands addressed for ADH genotyping. The lipid profile and occurrence of premature atherosclerotic cardiovascular diseases were compared between the LDLR+APOE carriers (n=21) and the carriers of the same LDLR causative variants alone (n=22). RESULTS: The prevalence of LDLR+APOE carriers in this French ADH cohort is 0.4%. Overall, LDL (low-density lipoprotein)-cholesterol concentrations were 23% higher in LDLR+APOE patients than in LDLR patients (9.14±2.51 versus 7.43±1.59 mmol/L, P=0.0221). When only deleterious or probably deleterious variants were considered, the LDL-cholesterol concentrations were 46% higher in LDLR+APOE carriers than in LDLR carriers (10.83±3.45 versus 7.43±1.59 mmol/L, P=0.0270). Two patients exhibited a homozygous familial hypercholesterolemia phenotype (LDL-cholesterol >13 mmol/L). Premature atherosclerotic cardiovascular disease was more common in LDLR+APOE patients than in LDLR carriers (70% versus 30%, P=0.026). CONCLUSIONS: Although an incomplete penetrance should be taken into account for APOE variant classification, these results suggest an additive effect of deleterious APOE variants on ADH phenotype highlighting the relevance of APOE sequencing.
Subject(s)
Atherosclerosis , Hyperlipoproteinemia Type II , Humans , Proprotein Convertase 9/genetics , Receptors, LDL/genetics , Hyperlipoproteinemia Type II/diagnosis , Hyperlipoproteinemia Type II/epidemiology , Hyperlipoproteinemia Type II/genetics , Cholesterol, LDL , Phenotype , Atherosclerosis/epidemiology , Atherosclerosis/genetics , Apolipoproteins B/genetics , Apolipoproteins E/genetics , Mutation , HeterozygoteABSTRACT
Primary hypercholesterolemia is characterized by elevated LDL-cholesterol (LDL-C) levels isolated in autosomal dominant hypercholesterolemia (ADH) or associated with elevated triglyceride levels in familial combined hyperlipidemia (FCHL). Rare APOE variants are known in ADH and FCHL. We explored the APOE molecular spectrum in a French ADH/FCHL cohort of 5743 unrelated probands. The sequencing of LDLR, PCSK9, APOB, and APOE revealed 76 carriers of a rare APOE variant, with no mutation in LDLR, PCSK9, or APOB. Among the 31 APOE variants identified here, 15 are described in ADH, 10 in FCHL, and 6 in both probands. Five were previously reported with dyslipidemia and 26 are novel, including 12 missense, 5 synonymous, 2 intronic, and 7 variants in regulatory regions. Sixteen variants were predicted as pathogenic or likely pathogenic, and their carriers had significantly lower polygenic risk scores (wPRS) than carriers of predicted benign variants. We observed no correlation between LDL-C levels and wPRS, suggesting a major effect of APOE variants. Carriers of p.Leu167del were associated with a severe phenotype. The analysis of 11 probands suggests that carriers of an APOE variant respond better to statins than carriers of a LDLR mutation. Altogether, we show that the APOE variants account for a significant contribution to ADH and FCHL.
Subject(s)
Apolipoproteins E , Hyperlipoproteinemia Type II , Proprotein Convertase 9 , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Cholesterol, LDL/genetics , Cholesterol, LDL/metabolism , Humans , Hyperlipoproteinemia Type II/genetics , Hyperlipoproteinemia Type II/metabolism , Proprotein Convertase 9/genetics , Proprotein Convertase 9/metabolismABSTRACT
Bone marrow (BM) failure (BMF) in children and young adults is often suspected to be inherited, but in many cases diagnosis remains uncertain. We studied a cohort of 179 patients (from 173 families) with BMF of suspected inherited origin but unresolved diagnosis after medical evaluation and Fanconi anemia exclusion. All patients had cytopenias, and 12.0% presented ≥5% BM blast cells. Median age at genetic evaluation was 11 years; 20.7% of patients were aged ≤2 years and 36.9% were ≥18 years. We analyzed genomic DNA from skin fibroblasts using whole-exome sequencing, and were able to assign a causal or likely causal germ line mutation in 86 patients (48.0%), involving a total of 28 genes. These included genes in familial hematopoietic disorders (GATA2, RUNX1), telomeropathies (TERC, TERT, RTEL1), ribosome disorders (SBDS, DNAJC21, RPL5), and DNA repair deficiency (LIG4). Many patients had an atypical presentation, and the mutated gene was often not clinically suspected. We also found mutations in genes seldom reported in inherited BMF (IBMF), such as SAMD9 and SAMD9L (N = 16 of the 86 patients, 18.6%), MECOM/EVI1 (N = 6, 7.0%), and ERCC6L2 (N = 7, 8.1%), each of which was associated with a distinct natural history; SAMD9 and SAMD9L patients often experienced transient aplasia and monosomy 7, whereas MECOM patients presented early-onset severe aplastic anemia, and ERCC6L2 patients, mild pancytopenia with myelodysplasia. This study broadens the molecular and clinical portrait of IBMF syndromes and sheds light on newly recognized disease entities. Using a high-throughput sequencing screen to implement precision medicine at diagnosis can improve patient management and family counseling.
Subject(s)
Bone Marrow Diseases/genetics , Germ-Line Mutation , Adolescent , Bone Marrow Diseases/epidemiology , Child , Child, Preschool , Cohort Studies , DNA Mutational Analysis , Female , High-Throughput Nucleotide Sequencing , Humans , Infant , Infant, Newborn , Male , Myelodysplastic Syndromes/epidemiology , Myelodysplastic Syndromes/genetics , Exome SequencingABSTRACT
The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34+ cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl-/- mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [125I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells.
Subject(s)
Cell Membrane/metabolism , Mutation/genetics , Receptors, Thrombopoietin/genetics , Thrombocytosis/genetics , Thrombocytosis/pathology , Animals , Cell Line , Disease Models, Animal , Endoplasmic Reticulum Stress , Family , Female , Humans , Male , Megakaryocytes/metabolism , Mice , Pedigree , Protein Transport , Receptors, Thrombopoietin/metabolism , Retroviridae/metabolism , Transduction, GeneticABSTRACT
Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN.
Subject(s)
Mutation , Receptors, Thrombopoietin/genetics , Thrombocythemia, Essential/genetics , Amino Acid Substitution , Cell Line , Cell Proliferation/drug effects , Cell Proliferation/genetics , Codon , Cohort Studies , Comparative Genomic Hybridization , Cytokines/pharmacology , DNA Mutational Analysis , Exome , Genotype , Granulocytes/metabolism , High-Throughput Nucleotide Sequencing , Humans , Janus Kinase 2/genetics , Protein Transport , Receptors, Thrombopoietin/metabolism , Thrombocythemia, Essential/metabolismABSTRACT
The transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.
Subject(s)
GATA1 Transcription Factor/metabolism , Ikaros Transcription Factor/physiology , Receptors, Notch/metabolism , Thrombopoiesis/genetics , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Proliferation , Cells, Cultured , Down-Regulation/genetics , Embryo, Mammalian , Gene Expression Regulation, Developmental , Ikaros Transcription Factor/genetics , Ikaros Transcription Factor/metabolism , Megakaryocytes/metabolism , Megakaryocytes/physiology , Mice , Mice, Knockout , Models, Biological , Protein Binding/genetics , Protein Binding/physiology , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
The intestinal epithelium is a complex system characterized by massive and continuous cell renewal and differentiation. In this context, cell-type-specific transcription factors are thought to play a crucial role by modulating specific transcription networks and signalling pathways. Hnf1alpha and beta are closely related atypical homeoprotein transcription factors expressed in several epithelia, including the gut. With the use of a conditional inactivation system, we generated mice in which Hnf1b is specifically inactivated in the intestinal epithelium on a wild-type or Hnf1a(-/-) genetic background. Whereas the inactivation of Hnf1a or Hnf1b alone did not lead to any major intestinal dysfunction, the concomitant inactivation of both genes resulted in a lethal phenotype. Double-mutant animals had defective differentiation and cell fate commitment. The expression levels of markers of all the differentiated cell types, both enterocytes and secretory cells, were affected. In addition, the number of goblet cells was increased, whereas mature Paneth cells were missing. At the molecular level, we show that Hnf1alpha and beta act upstream of the Notch pathway controlling directly the expression of two crucial components: Jag1 and Atoh1. We demonstrate that the double-mutant mice present with a defect in intestinal water absorption and that Hnf1alpha and beta directly control the expression of Slc26a3, a gene whose mutations are associated with chloride diarrhoea in human patients. Our study identifies new direct target genes of the Hnf1 transcription factors and shows that they play crucial roles in both defining cell fate and controlling terminal functions in the gut epithelium.
Subject(s)
Cell Differentiation/physiology , Hepatocyte Nuclear Factor 1-alpha/metabolism , Hepatocyte Nuclear Factor 1-beta/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Animals , Antiporters/genetics , Antiporters/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Differentiation/genetics , Chromatin Immunoprecipitation , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-beta/genetics , Immunohistochemistry , In Situ Hybridization , In Vitro Techniques , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Polymerase Chain Reaction , Serrate-Jagged Proteins , Sulfate TransportersABSTRACT
Transforming growth factor-ß1 (TGF-ß1) is the most important cytokine involved in the promotion of myelofibrosis. Mechanisms leading to its local activation in the bone marrow environment remain unclear. As a recent study has highlighted the role of thrombospondin-1 (TSP-1) in platelet-derived TGF-ß1 activation, we investigated the role of TSP-1 in the TPO(high) murine model of myelofibrosis. Two groups of engrafted mice, WT TPO(high) and Tsp-1-null TPO(high), were constituted. All mice developed a similar myeloproliferative syndrome and an increase in total TGF-ß1 levels in the plasma and in extracellular fluids of marrow and spleen. Surprisingly, we were able to detect the active form of TGF-ß1 in Tsp-1-null TPO(high) mice. Accordingly, these mice developed marrow and spleen fibrosis, with intriguingly a higher grade than in WT TPO(high) mice. Our results show that TSP-1 is not the major activator of TGF-ß1 in TPO-induced myelofibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment.
Subject(s)
Primary Myelofibrosis/chemically induced , Primary Myelofibrosis/pathology , Thrombopoietin/adverse effects , Thrombospondin 1/physiology , Transforming Growth Factor beta1/metabolism , Animals , Blood Platelets/metabolism , Blood Platelets/pathology , Bone Marrow/metabolism , Bone Marrow/pathology , Female , Male , Megakaryocytes/metabolism , Megakaryocytes/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Primary Myelofibrosis/metabolism , Spleen/metabolism , Spleen/pathologyABSTRACT
Liver adenomas are benign tumors at risk of malignant transformation. In a genome-wide search for loss of heterozygosity (LOH) associated with liver adenomas, we found a deletion in chromosome 12q in five of ten adenomas. In most cases, LOH at 12q was the only recurrent genetic alteration observed, suggesting the presence of a tumor-suppressor gene in that region. A minimal common region of deletion was defined in 12q24 that included the gene TCF1 (transcription factor 1), encoding hepatocyte nuclear factor 1 (HNF1; refs 1,2). Heterozygous germline mutations of TCF1 have been identified in individuals affected with maturity-onset diabetes of the young type 3 (MODY3; ref. 3). Bi-allelic inactivation of TCF1 was found in 10 of 16 screened adenomas, and heterozygous germline mutation were present in three affected individuals. Furthermore, 2 well-differentiated hepatocellular carcinomas (HCCs) occurring in normal liver contained somatic bi-allelic mutations of 30 screened HCCs. These results indicate that inactivation of TCF1, whether sporadic or associated with MODY3, is an important genetic event in the occurrence of human liver adenoma, and may be an early step in the development of some HCCs.
Subject(s)
Adenoma/genetics , DNA-Binding Proteins , Liver Neoplasms/genetics , Nuclear Proteins , Transcription Factors/genetics , Adenoma/physiopathology , Adult , Aged , Chromosomes, Human, Pair 12 , Diabetes Mellitus, Type 2/genetics , Female , Genetic Markers , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Humans , Liver Neoplasms/metabolism , Loss of Heterozygosity , Male , Microsatellite Repeats , Middle Aged , Mutation, Missense , Sequence Analysis, DNAABSTRACT
Fanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects.
Subject(s)
Fanconi Anemia , Leukemia , Humans , Mice , Animals , Fanconi Anemia/genetics , Clonal Hematopoiesis , Trisomy/genetics , Tumor Suppressor Protein p53/genetics , Leukemia/genetics , Chromosomes , Hematopoiesis/genetics , Proto-Oncogene Proteins/genetics , Cell Cycle Proteins/geneticsABSTRACT
Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.
Subject(s)
Calreticulin/adverse effects , Hematopoiesis/drug effects , Myeloproliferative Disorders/genetics , Female , Humans , Male , MutationABSTRACT
Frameshift mutations in the calreticulin (CALR) gene are present in 30% of essential thrombocythemia and myelofibrosis patients. The two most frequent mutations are CALR del52 (type 1, approximately 60%) and CALR ins5 (type 2, around 30%), but many other rarer mutations exist accounting each for less than 2% of all CALR mutations. Most of them are structurally classified as type 1-like and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha-helix structure. Yet, several key questions remain unanswered, especially the reason of such low frequencies of these other mutations. In an attempt to investigate specific pathogenic differences between type 1-like and type 2-like CALR mutations and del52 and ins5, we modeled two type 1-like (del34 and del46) and one type 2-like (del19) mutations in cell lines and in mice. All CALR mutants constitutively activate JAK2 and STAT5/3/1 in a similar way in the presence of the thrombopoietin receptor (MPL) and induced cytokine-independent cell growth but to a lesser extent with rare mutants over time. This correlates with reduced expression levels of rare CALR mutants compared to del52 and ins5. Lethally irradiated mice that were engrafted with bone marrow transduced with the different CALR mutations developed thrombocytosis, but to a much lesser extent with ins5 and the type 2-like CALR mutation. In contrast to type 2-like mice, type 1-like mice developed marked myelofibrosis and splenomegaly 10 months after engraftment. Similar to del52, type 1-like CALR mutations induced an expansion at an early stage of hematopoiesis compared to ins5 and type 2-like mutation. Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble del52 and ins5 mutants, respectively.
Subject(s)
Calreticulin/genetics , Calreticulin/metabolism , Mutation , Myeloproliferative Disorders/genetics , Animals , Calreticulin/chemistry , Cell Line , Disease Models, Animal , Humans , Janus Kinase 2/genetics , Mice , Myeloproliferative Disorders/metabolism , Receptors, Thrombopoietin/metabolism , STAT Transcription Factors/genetics , Transcriptional ActivationABSTRACT
RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.
Subject(s)
Dyskeratosis Congenita/genetics , Epigenomics/methods , Germ-Line Mutation , Nuclear Proteins/genetics , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Ribosomal/genetics , Animals , Dyskeratosis Congenita/pathology , Gene Expression Profiling , Hematopoietic Stem Cells , Male , Methylation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/chemistry , Nucleophosmin , RNA, Small Nucleolar , TranscriptomeABSTRACT
Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6-CTTN axis as a positive regulator of human but not mouse MK maturation.
Subject(s)
Cortactin/metabolism , Histone Deacetylase 6/metabolism , Megakaryocytes/metabolism , Thrombocytopenia/metabolism , Acetylation/drug effects , Animals , Blood Platelets/cytology , Blood Platelets/metabolism , Cell Differentiation/genetics , Cells, Cultured , Cortactin/genetics , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase 6/genetics , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Megakaryocytes/cytology , Mice, Knockout , Pyrimidines/pharmacology , RNA Interference , Thrombocytopenia/geneticsABSTRACT
Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.
Subject(s)
Fanconi Anemia/genetics , Mad2 Proteins/genetics , Mutation , Alleles , Animals , Cell Cycle , Cell Line, Tumor , Child , Chromosomal Instability , Chromosome Breakage , Cohort Studies , Cross-Linking Reagents/chemistry , DNA Damage , DNA Repair , Female , Fibroblasts/metabolism , Gene Silencing , Genetic Complementation Test , Genetic Predisposition to Disease , Genetic Variation , Hematopoietic Stem Cells/cytology , Humans , Lentivirus , Mad2 Proteins/metabolism , Male , Mice , Mice, Knockout , Mitosis , PhenotypeABSTRACT
One of the most frequent deletions in hepatocellular carcinoma (HCC) is that involving the long arm of chromosome 4 (30 to 70% of the cases). These chromosomal deletions are closely related to hepatitis B virus (HBV) infection. A tumor suppressor gene (TSG) located on 4q has been proposed in liver carcinogenesis, but has not been identified as yet. Despite previous LOH studies focused on 4q in HCC, a clear minimal common region of deletion (MCRD) could not be delimited. To further investigate the role of chromosome 4q LOH in the pathogenesis of HCC, 85 microsatellite markers spanning chromosome 4q were systematically analysed in a series of 154 well-characterized primary liver tumors. In 59 tumors (38%), LOHs were observed for at least two adjacent markers. Analysis of 31 tumors demonstrating a partial or interstitial 4q deletion allowed to define three MCRDs of 15, 9 and 8 Mb at the 4q22, 4q34 and 4q35 regions, respectively. Seven putative candidate genes located in 4q22, DAPP1, BMPR1B, PKD2, HERC3, SMARCAD1, CEB1 and ENH were screened for mutations but no somatic alterations were identified. Search for relationships between the specific regions of deletion and clinical parameters showed a significant association between loss of the 4q34-35 region with alcohol intake (P=0.005) and with high grade of differentiation (P=0.02). These results are in contrast with the close association between HBV infection and the whole 4q LOH and reveal heterogeneity of 4q LOH in relation to different risk factors. In the light of these new findings, which link different 4q LOH regions to different etiologic factors, the molecular mechanisms underlying 4q deletions in HCC and the targeted gene(s) remain to be identified.
Subject(s)
Alleles , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Differentiation/drug effects , Chromosome Deletion , Chromosomes, Human, Pair 4/genetics , Ethanol/pharmacology , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/etiology , Cell Line , DNA Mutational Analysis , Humans , Loss of Heterozygosity/genetics , Physical Chromosome MappingABSTRACT
Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57(Kip2) and p19(INK4d). Using the p19(INK4d-/-) mouse model, we investigated the role of p19(INK4d) in basal and stress-induced hematopoiesis. We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19(INK4d) in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19(INK4d) controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor ß1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.
Subject(s)
Cellular Senescence/genetics , Cyclin-Dependent Kinase Inhibitor p19/genetics , DNA Damage , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Stem Cell Niche/genetics , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Count , Cell Differentiation/genetics , Cell Lineage/genetics , Cyclin-Dependent Kinase Inhibitor p19/deficiency , Cyclin-Dependent Kinase Inhibitor p19/metabolism , Hematopoiesis , Mice , Mice, Knockout , Osteosclerosis/genetics , Osteosclerosis/pathology , Primary Myelofibrosis/genetics , Primary Myelofibrosis/pathology , Resting Phase, Cell Cycle/genetics , Stromal Cells/metabolismABSTRACT
Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.