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1.
Haematologica ; 2023 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-38152053

RESUMO

Mutations in five canonical Ras pathway genes (NF1, NRAS, KRAS, PTPN11 and CBL) are detected in nearly 90% of patients with juvenile myelomonocytic leukemia (JMML), a frequently fatal malignant neoplasm of early childhood. In this report, we describe seven patients diagnosed with SH2B3-mutated JMML, including five patients who were found to have initiating, loss of function mutations in the gene. SH2B3 encodes the adaptor protein LNK, a negative regulator of normal hematopoiesis upstream of the Ras pathway. These mutations were identified to be germline, somatic or a combination of both. Loss of function of LNK, which has been observed in other myeloid malignancies, results in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of the JAK/STAT signaling pathway. In vitro studies of induced pluripotent stem cell-derived JMML-like hematopoietic progenitor cells (HPCs) also demonstrated sensitivity of SH2B3- mutated HPCs to JAK inhibition. Lastly, we describe two patients with JMML and SH2B3 mutations who were treated with the JAK1/2 inhibitor ruxolitinib. This report expands the spectrum of initiating mutations in JMML and raises the possibility of targeting the JAK/STAT pathway in patients with SH2B3 mutations.

2.
Clin Genet ; 103(1): 109-113, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36075864

RESUMO

Prior studies have demonstrated that patients with chromosome 22q11.2 deletion syndrome (22q11.2DS) have lower platelet counts (PC) compared to non-deleted populations. They also have an increased mean platelet volume. The mechanism for this has been postulated to be haploinsufficiency of the GPIBB gene. We examined platelet parameters, deletion size and factors known to influence counts, including status of thyroid hormone and congenital heart disease (CHD), in a population of 825 patients with 22q11.2DS. We also measured surface expression of GPIB-IX complex by flow cytometry. The major determinant of PC was deletion status of GP1BB, regardless of surface expression or other factors. Patients with nested distal chromosome 22q11.2 deletions (those with GP1BB present) had higher PCs than those with proximal deletions where GP1BB is deleted. Patients with 22q11.2DS also demonstrated an accelerated PC decrease with age, occurring in childhood. These data demonstrate that genes within the proximal deletion segment drive PC differences in 22q11.2DS and suggest that PC reference ranges may need to be adjusted for age and deletion size in 22q11.2DS populations. Bleeding did not correlate with either platelet count or GPIb expression. Further studies into drivers of expression of GPIb and associations with severe thrombocytopenia and immune thrombocytopenia are needed to inform clinical care.


Assuntos
Síndrome de DiGeorge , Humanos , Síndrome de DiGeorge/genética
3.
J Clin Invest ; 132(19)2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-35925681

RESUMO

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.


Assuntos
Megacariócitos , Trombocitopenia , Actinas/metabolismo , Plaquetas/metabolismo , Humanos , Recém-Nascido , Megacariócitos/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Trombocitopenia/genética , Trombopoese/genética , Quinases Dyrk
4.
Epigenetics ; 16(12): 1295-1305, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33300436

RESUMO

Genomic imprinting is a rare form of gene expression in mammals in which a small number of genes are expressed in a parent-of-origin-specific manner. The aetiology of human imprinting disorders is diverse and includes chromosomal abnormalities, mutations, and epigenetic dysregulation of imprinted genes. The most common human imprinting disorder is Beckwith-Wiedemann syndrome (BWS), frequently caused by uniparental isodisomy and DNA methylation alterations. Because these lesions cannot be easily engineered, induced pluripotent stem cells (iPSC) are a compelling alternative. Here, we describe the first iPSC model derived from patients with BWS. Due to the mosaic nature of BWS patients, both BWS and non-BWS iPSC lines were derived from the same patient's fibroblasts. Importantly, we determine that DNA methylation and gene expression patterns of the imprinted region in the iPSC lines reflect the parental cells and are stable over time. Additionally, we demonstrate that differential expression in insulin signalling, cell proliferation, and cell cycle pathways was seen in hepatocyte lineages derived from BWS lines compared to controls. Thus, this cell based-model can be used to investigate the role of imprinting in the pathogenesis of BWS in disease-relevant cell types.


Assuntos
Síndrome de Beckwith-Wiedemann , Síndrome de Beckwith-Wiedemann/genética , Metilação de DNA , Impressão Genômica , Humanos , Mutação
5.
Stem Cell Res ; 49: 102084, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33202304

RESUMO

The CHOPWT4 iPSC line was generated as a control for applications such as differentiation analyses to the three germ layers and derivative tissues. Human foreskin fibroblasts were reprogrammed using the non-integrating Sendai virus expressing Oct3/4, Sox2, c-myc, and Klf4.


Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Células Epiteliais , Fibroblastos , Prepúcio do Pênis , Humanos , Fator 4 Semelhante a Kruppel , Masculino
6.
BMC Biol ; 18(1): 52, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32408895

RESUMO

BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits. RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields. CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.


Assuntos
Plaquetas/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Tropomiosina/metabolismo , Sistemas CRISPR-Cas , Estudo de Associação Genômica Ampla , Humanos , Técnicas In Vitro , Tropomiosina/deficiência
7.
Cell Stem Cell ; 25(2): 273-289.e5, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374199

RESUMO

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Humanas/fisiologia , Pâncreas/patologia , Respiração Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Proteínas do Leite , Mutação/genética , Pâncreas/fisiologia , Fenótipo , RNA Longo não Codificante/genética
8.
Stem Cell Reports ; 8(3): 589-604, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28196690

RESUMO

Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into ß-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient's disease. GATA6 mutant ß-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and ß-like cell functionality in humans.


Assuntos
Endoderma/metabolismo , Fator de Transcrição GATA6/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Endoderma/efeitos dos fármacos , Endoderma/embriologia , Fator de Transcrição GATA6/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Modelos Biológicos , Família Multigênica , Mutação , Pâncreas/embriologia , Fenótipo , Tretinoína/farmacologia
9.
Stem Cell Res ; 16(2): 338-41, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27345999

RESUMO

The CHOPWT10 iPS cell line was generated to be used as a control for applications such as in differentiation analyses to the three germ layers and derivative tissues. Peripheral blood mononuclear cells (PBMCs) obtained from a healthy adult male were reprogrammed using the non-integrating Sendai virus expressing Oct3/4, Sox2, c-Myc, and Klf4.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Leucócitos Mononucleares/citologia , Adulto , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Corpos Embrioides/citologia , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Fator 4 Semelhante a Kruppel , Masculino , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Hum Mol Genet ; 25(22): 4939-4950, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28171541

RESUMO

Substantial variability exists in the presentation of complex neurological disorders, and the study of single nucleotide polymorphisms (SNPs) has shed light on disease mechanisms and pathophysiological variability in some cases. However, the vast majority of disease-linked SNPs have unidentified pathophysiological relevance. Here, we tested the hypothesis that SNPs within the miRNA recognition element (MRE; the region of the target transcript to which the miRNA binds) can impart changes in the expression of those genes, either by enhancing or reducing transcript and protein levels. To test this, we cross-referenced 7,153 miRNA-MRE brain interactions with the SNP database (dbSNP) to identify candidates, and functionally assessed 24 SNPs located in the 3'UTR or the coding sequence (CDS) of targets. For over half of the candidates tested, SNPs either enhanced (4 genes) or disrupted (10 genes) miRNA binding and target regulation. Additionally, SNPs causing a shift from a common to rare codon within the CDS facilitated miRNA binding downstream of the SNP, dramatically repressing target gene expression. The biological activity of the SNPs on miRNA regulation was also confirmed in induced pluripotent stem cell (iPSC) lines. These studies strongly support the notion that SNPs in the 3'UTR or the coding sequence of disease-relevant genes may be important in disease pathogenesis and should be reconsidered as candidate modifiers.


Assuntos
Encéfalo/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Regiões 3' não Traduzidas , Sítios de Ligação , Encéfalo/metabolismo , Bases de Dados Genéticas , Células HEK293 , Humanos , MicroRNAs/metabolismo , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , Motivos de Ligação ao RNA
11.
Cell Stem Cell ; 18(1): 73-78, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26607381

RESUMO

Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.


Assuntos
Eritrócitos/citologia , Células-Tronco/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Citocinas/metabolismo , Células-Tronco Embrionárias/citologia , Sangue Fetal/citologia , Técnicas Genéticas , Variação Genética , Genoma Humano , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/análise , Humanos , Mutação , Células-Tronco Pluripotentes/citologia , Medicina Regenerativa/métodos
12.
Blood ; 127(6): 675-80, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26634302

RESUMO

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin ß3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.


Assuntos
Antígenos de Plaquetas Humanas/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Isoantígenos/genética , Antígenos de Plaquetas Humanas/imunologia , Sequência de Bases , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Integrina beta3/genética , Integrina beta3/imunologia , Isoanticorpos/genética , Isoanticorpos/imunologia , Isoantígenos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Polimorfismo de Nucleotídeo Único
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