RESUMO
Colorectal malignancies are a leading cause of cancer-related death1 and have undergone extensive genomic study2,3. However, DNA mutations alone do not fully explain malignant transformation4-7. Here we investigate the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,370 samples from 30 primary cancers and 8 concomitant adenomas and generated 1,207 chromatin accessibility profiles, 527 whole genomes and 297 whole transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent somatic chromatin accessibility alterations, including in regulatory regions of cancer driver genes that were otherwise devoid of genetic mutations. Genome-wide alterations in accessibility for transcription factor binding involved CTCF, downregulation of interferon and increased accessibility for SOX and HOX transcription factor families, suggesting the involvement of developmental genes during tumourigenesis. Somatic chromatin accessibility alterations were heritable and distinguished adenomas from cancers. Mutational signature analysis showed that the epigenome in turn influences the accumulation of DNA mutations. This study provides a map of genetic and epigenetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.
Assuntos
Neoplasias Colorretais , Epigenoma , Genoma Humano , Mutação , Humanos , Adenoma/genética , Adenoma/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/genética , Cromatina/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Epigenoma/genética , Oncogenes/genética , Fatores de Transcrição/metabolismo , Genoma Humano/genética , InterferonsRESUMO
Genetic and epigenetic variation, together with transcriptional plasticity, contribute to intratumour heterogeneity1. The interplay of these biological processes and their respective contributions to tumour evolution remain unknown. Here we show that intratumour genetic ancestry only infrequently affects gene expression traits and subclonal evolution in colorectal cancer (CRC). Using spatially resolved paired whole-genome and transcriptome sequencing, we find that the majority of intratumour variation in gene expression is not strongly heritable but rather 'plastic'. Somatic expression quantitative trait loci analysis identified a number of putative genetic controls of expression by cis-acting coding and non-coding mutations, the majority of which were clonal within a tumour, alongside frequent structural alterations. Consistently, computational inference on the spatial patterning of tumour phylogenies finds that a considerable proportion of CRCs did not show evidence of subclonal selection, with only a subset of putative genetic drivers associated with subclone expansions. Spatial intermixing of clones is common, with some tumours growing exponentially and others only at the periphery. Together, our data suggest that most genetic intratumour variation in CRC has no major phenotypic consequence and that transcriptional plasticity is, instead, widespread within a tumour.
Assuntos
Adaptação Fisiológica , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Fenótipo , Humanos , Adaptação Fisiológica/genética , Células Clonais/metabolismo , Células Clonais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Sequenciamento do Exoma , Transcrição GênicaRESUMO
Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions.
Assuntos
Linfócitos/imunologia , MicroRNAs/imunologia , Animais , Células HEK293 , Homeostase , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genéticaRESUMO
EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.
Assuntos
Anormalidades Musculoesqueléticas/genética , N-Acetilglucosaminiltransferases/genética , Osteocondrodisplasias/genética , Alelos , Linhagem Celular , Linhagem Celular Tumoral , Condroitina/sangue , Condroitina/urina , Variações do Número de Cópias de DNA , Estudo de Associação Genômica Ampla , Glicosaminoglicanos/metabolismo , Humanos , Anormalidades Musculoesqueléticas/diagnóstico , Mutação de Sentido Incorreto , Osteocondrodisplasias/diagnóstico , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/genéticaRESUMO
Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.
Assuntos
Ataxia Cerebelar/genética , Deficiência Intelectual/genética , Nexinas de Classificação/genética , Sequência de Bases , Ataxia Cerebelar/patologia , Mapeamento Cromossômico , Códon sem Sentido/genética , Feminino , Fibroblastos/ultraestrutura , Redes Reguladoras de Genes/genética , Genes Recessivos/genética , Humanos , Deficiência Intelectual/patologia , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Mutations in SLC25A22 are known to cause neonatal epileptic encephalopathy and migrating partial seizures in infancy. Using whole exome sequencing we identified four novel SLC25A22 mutations in six children from three families. Five patients presented clinical features similar to those in the literature including hypotonia, refractory neonatal-onset seizures and developmental delay. However, the sixth patients presented atypically with isolated developmental delay, developing late-onset (absence) seizures only at 7 years of age. Abnormal metabolite levels have not been documented in the nine patients described previously. One patient in our series was referred to the metabolic clinic because of persistent hyperprolinaemia and another three had raised plasma proline when tested. Analysis of the post-prandial plasma amino acid response in one patient showed abnormally high concentrations of several amino acids. This suggested that, in the fed state, when amino acids are the preferred fuel for the liver, trans-deamination of amino acids requires transportation of glutamate into liver mitochondria by SLC25A22 for deamination by glutamate dehydrogenase; SLC25A22 is an important mitochondrial glutamate transporter in liver as well as in brain. Electron microscopy of patient fibroblasts demonstrated widespread vacuolation containing neutral and phospho-lipids as demonstrated by Oil Red O and Sudan Black tinctorial staining; this might be explained by impaired activity of the proline/pyrroline-5-carboxylate (P5C) shuttle if SLC25A22 transports pyrroline-5-carboxylate/glutamate-γ-semialdehyde as well as glutamate.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Deficiências do Desenvolvimento/genética , Fibroblastos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Mutação/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Criança , Pré-Escolar , Feminino , Ácido Glutâmico/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/metabolismo , Hipotonia Muscular/genética , Hipotonia Muscular/metabolismo , Prolina/metabolismo , Convulsões/genética , Convulsões/metabolismoRESUMO
BACKGROUND: Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. DESIGN: We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). RESULTS: TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. CONCLUSIONS: Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças Inflamatórias Intestinais/genética , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de DNA/métodos , Idade de Início , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos ProspectivosRESUMO
BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families. METHODS AND RESULTS: Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patient's respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD. CONCLUSIONS: We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.
Assuntos
Proteínas do Domínio Armadillo/genética , Dineínas/genética , Estudo de Associação Genômica Ampla , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Mutação , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Cílios/genética , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/química , Dineínas/metabolismo , Exoma , Feminino , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Cancer evolution lays the groundwork for predictive oncology. Testing evolutionary metrics requires quantitative measurements in controlled clinical trials. We mapped genomic intratumor heterogeneity in locally advanced prostate cancer using 642 samples from 114 individuals enrolled in clinical trials with a 12-year median follow-up. We concomitantly assessed morphological heterogeneity using deep learning in 1,923 histological sections from 250 individuals. Genetic and morphological (Gleason) diversity were independent predictors of recurrence (hazard ratio (HR) = 3.12 and 95% confidence interval (95% CI) = 1.34-7.3; HR = 2.24 and 95% CI = 1.28-3.92). Combined, they identified a group with half the median time to recurrence. Spatial segregation of clones was also an independent marker of recurrence (HR = 2.3 and 95% CI = 1.11-4.8). We identified copy number changes associated with Gleason grade and found that chromosome 6p loss correlated with reduced immune infiltration. Matched profiling of relapse, decades after diagnosis, confirmed that genomic instability is a driving force in prostate cancer progression. This study shows that combining genomics with artificial intelligence-aided histopathology leads to the identification of clinical biomarkers of evolution.
RESUMO
Patients with estrogen receptor-positive breast cancer receive adjuvant endocrine therapies (ET) that delay relapse by targeting clinically undetectable micrometastatic deposits. Yet, up to 50% of patients relapse even decades after surgery through unknown mechanisms likely involving dormancy. To investigate genetic and transcriptional changes underlying tumor awakening, we analyzed late relapse patients and longitudinally profiled a rare cohort treated with long-term neoadjuvant ETs until progression. Next, we developed an in vitro evolutionary study to record the adaptive strategies of individual lineages in unperturbed parallel experiments. Our data demonstrate that ETs induce nongenetic cell state transitions into dormancy in a stochastic subset of cells via epigenetic reprogramming. Single lineages with divergent phenotypes awaken unpredictably in the absence of recurrent genetic alterations. Targeting the dormant epigenome shows promising activity against adapting cancer cells. Overall, this study uncovers the contribution of epigenetic adaptation to the evolution of resistance to ETs. SIGNIFICANCE: This study advances the understanding of therapy-induced dormancy with potential clinical implications for breast cancer. Estrogen receptor-positive breast cancer cells adapt to endocrine treatment by entering a dormant state characterized by strong heterochromatinization with no recurrent genetic changes. Targeting the epigenetic rewiring impairs the adaptation of cancer cells to ETs. See related commentary by Llinas-Bertran et al., p. 704. This article is featured in Selected Articles from This Issue, p. 695.
Assuntos
Neoplasias da Mama , Epigênese Genética , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Recidiva Local de Neoplasia/genética , Regulação Neoplásica da Expressão GênicaRESUMO
The dominant mutational signature in colorectal cancer genomes is C > T deamination (COSMIC Signature 1) and, in a small subgroup, mismatch repair signature (COSMIC signatures 6 and 44). Mutations in common colorectal cancer driver genes are often not consistent with those signatures. Here we perform whole-genome sequencing of normal colon crypts from cancer patients, matched to a previous multi-omic tumour dataset. We analyse normal crypts that were distant vs adjacent to the cancer. In contrast to healthy individuals, normal crypts of colon cancer patients have a high incidence of pks + (polyketide synthases) E.coli (Escherichia coli) mutational and indel signatures, and this is confirmed by metagenomics. These signatures are compatible with many clonal driver mutations detected in the corresponding cancer samples, including in chromatin modifier genes, supporting their role in early tumourigenesis. These results provide evidence that pks + E.coli is a potential driver of carcinogenesis in the human gut.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Humanos , Escherichia coli/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Carcinogênese/genéticaRESUMO
In cancer, evolutionary forces select for clones that evade the immune system. Here we analyzed >10,000 primary tumors and 356 immune-checkpoint-treated metastases using immune dN/dS, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome, to measure immune selection in cohorts and individuals. We classified tumors as immune edited when antigenic mutations were removed by negative selection and immune escaped when antigenicity was covered up by aberrant immune modulation. Only in immune-edited tumors was immune predation linked to CD8 T cell infiltration. Immune-escaped metastases experienced the best response to immunotherapy, whereas immune-edited patients did not benefit, suggesting a preexisting resistance mechanism. Similarly, in a longitudinal cohort, nivolumab treatment removes neoantigens exclusively in the immunopeptidome of nonimmune-edited patients, the group with the best overall survival response. Our work uses dN/dS to differentiate between immune-edited and immune-escaped tumors, measuring potential antigenicity and ultimately helping predict response to treatment.
Assuntos
Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Nivolumabe , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos , MutaçãoRESUMO
Hyperinsulinaemic hypoglycaemia (HH) is due to the unregulated secretion of insulin from pancreatic ß-cells. A rapid diagnosis and appropriate management of these patients is essential to prevent the potentially associated complications like epilepsy, cerebral palsy and neurological impairment. The molecular basis of HH involves defects in key genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A and UCP2) which regulate insulin secretion. The most severe forms of HH are due to loss of function mutations in ABCC8/KCNJ11 which encode the SUR1 and KIR6.2 components respectively of the pancreatic ß-cell K(ATP) channel. At a histological level there are two major forms (diffuse and focal) each with a different genetic aetiology. The diffuse form is inherited in an autosomal recessive (or dominant) manner whereas the focal form is sporadic in inheritance and is localised to a small region of the pancreas. The focal form can now be accurately localised pre-operatively using a specialised positron emission tomography scan with the isotope Fluroine-18L-3, 4-dihydroxyphenyalanine (18F-DOPA-PET). Focal lesionectomy can provide cure from the hypoglycaemia. However the diffuse form is managed medically or by near total pancreatectomy (with high risk of diabetes mellitus). Recent advances in molecular genetics, imaging with 18F-DOPA-PET/CT and novel surgical techniques have changed the clinical approach to patients with HH.
Assuntos
Hiperinsulinismo Congênito/diagnóstico , Hiperinsulinismo Congênito/genética , Animais , Hiperinsulinismo Congênito/patologia , Hiperinsulinismo Congênito/terapia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Imagem Multimodal/métodos , Pancreatectomia/métodos , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Primary ovarian insufficiency (POI) affects 1% of women and is associated with significant medical consequences. A genetic cause for POI can be found in up to 30% of women, elucidating key roles for these genes in human ovary development. OBJECTIVE: We aimed to identify the genetic mechanism underlying early-onset POI in 2 sisters from a consanguineous pedigree. METHODS: Genome sequencing and variant filtering using an autosomal recessive model was performed in the 2 affected sisters and their unaffected family members. Quantitative reverse transcriptase PCR (qRT-PCR) and RNA sequencing were used to study the expression of key genes at critical stages of human fetal gonad development (Carnegie Stage 22/23, 9 weeks post conception (wpc), 11 wpc, 15/16 wpc, 19/20 wpc) and in adult tissue. RESULTS: Only 1 homozygous variant cosegregating with the POI phenotype was found: a single nucleotide substitution in zinc finger SWIM-type containing 7 (ZSWIM7), NM_001042697.2: c.173Câ >â G; resulting in predicted loss-of-function p.(Ser58*). qRT-PCR demonstrated higher expression of ZSWIM7 in the 15/16 wpc ovary compared with testis, corresponding to peak meiosis in the fetal ovary. RNA sequencing of fetal gonad samples showed that ZSWIM7 has a similar temporal expression profile in the developing ovary to other homologous recombination genes. MAIN CONCLUSIONS: Disruption of ZSWIM7 is associated with POI in humans. ZSWIM7 is likely to be important for human homologous recombination; these findings expand the range of genes associated with POI in women.
Assuntos
Amenorreia/genética , Proteínas de Ligação a DNA/genética , Meiose/genética , Oogênese/genética , Insuficiência Ovariana Primária/genética , Adolescente , Amenorreia/diagnóstico , Criança , Análise Mutacional de DNA , Feminino , Humanos , Mutação com Perda de Função , Ovário/crescimento & desenvolvimento , Linhagem , Mutação Puntual , Insuficiência Ovariana Primária/complicações , Insuficiência Ovariana Primária/diagnóstico , RNA-Seq , Dedos de ZincoRESUMO
OBJECTIVE: Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. METHODS: To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. RESULTS: Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89-0.95]) and reproducible (>0.87 [95% CI: 0.87-0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. CONCLUSIONS: This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias , Adulto , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , Criança , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologia , Sequenciamento Completo do Genoma/métodosRESUMO
Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.
Assuntos
Subunidade alfa de Receptor de Interleucina-7/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Células Precursoras de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos CD34/genética , Antígenos CD34/imunologia , Antígenos CD34/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Expressão Gênica/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfócitos B/metabolismo , RNA-Seq/métodos , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , Receptores de Citocinas/metabolismo , Transdução de Sinais/genética , Análise de Célula Única/métodos , Transplante HeterólogoRESUMO
Pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID) syndrome is a recently described autosomal recessive disorder associated with predominantly antibody negative, insulin-dependent diabetes mellitus. In order to identify the genetic basis of PHID and study its relationship with glucose metabolism, we performed homozygosity mapping in five unrelated families followed by candidate gene sequencing. Five loss-of-function mutations were identified in the SLC29A3 gene which encodes a member of a highly conserved protein family that transports nucleosides, nucleobases and nucleoside analogue drugs, hENT3. We show that PHID is allelic with a related syndrome without diabetes mellitus, H syndrome. The interaction of SLC29A3 with insulin signaling pathways was then studied using an established model in Drosophila melanogaster. Ubiquitous knockdown of the Drosophila ortholog of hENT3, dENT1 is lethal under stringent conditions; whereas milder knockdown induced scutellar bristle phenotypes similar to those previously reported in the knockdown of the Drosophila ortholog of the Islet gene. A cellular growth assay showed a reduction of cell size/number which could be rescued or enhanced by manipulation of the Drosophila insulin receptor and its downstream signaling effectors, dPI3K and dAkt. In summary, inactivating mutations in SLC29A3 cause a syndromic form of insulin-dependent diabetes in humans and in Drosophila profoundly affect cell size/number through interactions with the insulin signaling pathway. These data suggest that further investigation of the role of SLC29A3 in glucose metabolism is a priority for diabetes research.
Assuntos
Diabetes Mellitus Tipo 1/genética , Hipertricose/genética , Insulina/metabolismo , Mutação , Proteínas de Transporte de Nucleosídeos/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Humanos , Hipertricose/metabolismo , Insulina/genética , Masculino , Dados de Sequência Molecular , Proteínas de Transporte de Nucleosídeos/química , Proteínas de Transporte de Nucleosídeos/metabolismo , Linhagem , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Alinhamento de Sequência , Pigmentação da PeleRESUMO
Permanent neonatal diabetes mellitus (PNDM) caused by homozygous mutations in the glucokinase gene (GCK) is rare and only eight homozygous GCK mutations have been reported so far. Heterozygous GCK mutations cause maturity-onset diabetes of the young (MODY). We report four patients with growth retardation from two separate families (with three siblings in one family and one patient in another family) presenting with persistent hyperglycaemia within the first two days of life. We found one homozygous non-sense mutation (Q98X) in GCK in three siblings from one family and a homozygous missense GCK mutation (G261R) in one patient from another family. Both mutations have been identified previously in GCK-MODY in the heterozygous state. However, this is the first study to report the homozygous forms of these mutations in PNDM. We report four novel cases of PNDM caused by homozygous GCK mutations, including a non-sense mutation in exon 3 (Q98X) and a missense mutation in exon 7 (G261R).
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Glucoquinase/genética , Doenças do Recém-Nascido/genética , Códon sem Sentido , Feminino , Homozigoto , Humanos , Recém-Nascido , Masculino , Mutação de Sentido Incorreto , Linhagem , IrmãosRESUMO
Wolfram syndrome is a progressive neurodegenerative disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness). The majority of cases are caused by mutations in the WFS1 gene. WFS1 is located at 4p16.1 and encodes wolframin, a transmembrane endoplasmic reticulum (ER) protein involved in the negative regulation of ER stress signalling. To date, over 120 WFS1 mutations have been described. In this study, we report a consanguineous family with three siblings affected by Wolfram syndrome. A homozygous single base pair deletion (c.877delC, L293fsX303) was found in the WFS1 gene in all three affected siblings.
Assuntos
Proteínas de Membrana/genética , Deleção de Sequência , Síndrome de Wolfram/genética , Adolescente , Sequência de Bases , Criança , Códon sem Sentido , Consanguinidade , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Homozigoto , Humanos , Masculino , Linhagem , Irmãos , Síndrome de Wolfram/metabolismo , Adulto JovemRESUMO
Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs in vivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.