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Transcriptomics is a powerful tool for unraveling the molecular effects of genetic variants and disease diagnosis. Prior studies have demonstrated that choice of genome build impacts variant interpretation and diagnostic yield for genomic analyses. To identify the extent genome build also impacts transcriptomics analyses, we studied the effect of the hg19, hg38, and CHM13 genome builds on expression quantification and outlier detection in 386 rare disease and familial control samples from both the Undiagnosed Diseases Network and Genomics Research to Elucidate the Genetics of Rare Disease Consortium. Across six routinely collected biospecimens, 61% of quantified genes were not influenced by genome build. However, we identified 1,492 genes with build-dependent quantification, 3,377 genes with build-exclusive expression, and 9,077 genes with annotation-specific expression across six routinely collected biospecimens, including 566 clinically relevant and 512 known OMIM genes. Further, we demonstrate that between builds for a given gene, a larger difference in quantification is well correlated with a larger change in expression outlier calling. Combined, we provide a database of genes impacted by build choice and recommend that transcriptomics-guided analyses and diagnoses are cross referenced with these data for robustness.
Assuntos
Genoma Humano , RNA-Seq , Humanos , RNA-Seq/métodos , Genômica/métodos , Transcriptoma , Doenças Raras/genética , Doenças Raras/diagnóstico , Perfilação da Expressão Gênica/métodosRESUMO
A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.
Assuntos
Proteína C9orf72/genética , Microbioma Gastrointestinal/fisiologia , Gliose/microbiologia , Gliose/patologia , Inflamação/genética , Inflamação/microbiologia , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Antibacterianos/farmacologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/genética , Autoimunidade/imunologia , Movimento Celular/efeitos dos fármacos , Citocinas/imunologia , Transplante de Microbiota Fecal , Feminino , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Gliose/genética , Gliose/prevenção & controle , Inflamação/patologia , Inflamação/prevenção & controle , Mutação com Perda de Função/genética , Masculino , Camundongos , Microglia/imunologia , Microglia/microbiologia , Microglia/patologia , Medula Espinal/imunologia , Medula Espinal/microbiologia , Taxa de SobrevidaRESUMO
PURPOSE: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in 4 siblings. METHODS: We identified 5 individuals from 3 unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort. RESULTS: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA sequencing and metabolomic data sets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation. CONCLUSION: Our data shed light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.
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BACKGROUND: Complete unilateral cleft lip and palate (UCLP) creates a continuity defect on the nasal floor, which contributes to nasal asymmetry. Traditionally, piriform rim symmetry has been evaluated by comparing cleft and noncleft sides. No study has compared the magnitude of perinasal asymmetry in UCLP patients with a control group of patients without clefts. PURPOSE: To address the following question: In UCLP patients, whose alveolar clefts are reconstructed with alveolar bone grafts (ABGs), is the magnitude of remaining piriform rim asymmetry similar to that of patients without UCLP? STUDY DESIGN SETTING, SAMPLE: This is a retrospective cohort study that used the cone beam computed tomography of UCLP and non-UCLP patients to evaluate the piriform rim symmetry. The sample was derived from patients who presented for orthognathic surgery between January 2015 and December 2022. To be included, patients had to have a maxillary deficiency. The cleft group had ABG performed with symphyseal bone harvest and bone morphogenetic protein application. Patients were excluded from the control group if they had clinical asymmetry and nasal septum deviation. Patients from the UCLP group were excluded if they failed the first attempt of ABG or had a syndrome. Preorthognathic cone beam computed tomography was used to measure the distance from the inferior and lateral aspects of the piriform rim to reference lines. PREDICTOR VARIABLE: UCLP status grouped as present or absent (control). OUTCOME VARIABLES: The magnitude of piriform rim asymmetry defined as the millimetric distance from the inferior and lateral aspects of the piriform rim to reference lines. COVARIATES: The covariates were age, sex, tissue thickness at the level of the alar base, and turbinate size. ANALYSIS: Welch's two-sample t-test was utilized to compare means. A level of significance of 5% (P < .05) was used for all analyses. To analyze the reliability of the measurements intraexaminer and interexaminer errors were tested using the Weir method. RESULTS: A total of 60 patients were included, 30 in each group. The mean age of UCLP patients was 16.76 (range 13 to 25), and the control group was 17 (range 13 to 25), P = .71. The UCLP group had 12 girls, and the control had 18 girls (P = .12). In the UCLP group, the mean discrepancy between affected and unaffected sides at the inferior aspect of the piriform rim was 3.9 mm (range 0.9 to 7 mm, P < .01), and in the control group the discrepancy between right and left sides was 0.1 mm (0-2.1 mm, P = .87). The mean discrepancy between affected and unaffected sides at the lateral aspect of the piriform rim was 3.6 mm (range 0.7 to 7.6 mm, P < .01) in the UCLP group, and in the control group the discrepancy between right and left sides was 0.1 mm (range 0.1 to 5.8 mm, P = .78) in the control group. The mean alar base soft tissue thickness discrepancy was 3.1 mm (range 0.9 to 7.9 mm, P < .01) in the UCLP group and 0 mm (range -1.8 to 1.9 mm, P = .97) in the control group. The mean difference in the turbinate area in the UCLP group was 314 mm2 (range 797 to 2,898) and in the control group 35 mm2 (range 702 to 2,302) (P = .19). CONCLUSION: ABG with symphyseal bone and bone morphogenetic protein was not able to provide the same level of piriform symmetry observed in patients without a cleft. Alar base tissue was thicker on the cleft side, and the turbinate size demonstrated greater variability in the UCLP patients.
Assuntos
Fenda Labial , Fissura Palatina , Cirurgia Ortognática , Feminino , Humanos , Fenda Labial/diagnóstico por imagem , Fenda Labial/cirurgia , Fissura Palatina/diagnóstico por imagem , Fissura Palatina/cirurgia , Estudos Retrospectivos , Reprodutibilidade dos Testes , Septo Nasal , Tomografia Computadorizada de Feixe Cônico/métodosRESUMO
This case report presents a palatal cleft that healed spontaneously, with complete formation of mucosa and bone. Even though the nasal structures could initially be observed through the cleft palate, a thin membrane sealed any communication between the oral and nasal cavities. The origin of this tenuous membrane cannot be fully understood with current discernment of palate formation, but it probably served as a basis for the formation of the other tissues. No previous record of nonintervened spontaneous closure of a cleft palate has been reported.
Assuntos
Fissura Palatina , Fissura Palatina/cirurgia , Humanos , Cavidade NasalRESUMO
The X Chromosome, with its unique mode of inheritance, contributes to differences between the sexes at a molecular level, including sex-specific gene expression and sex-specific impact of genetic variation. Improving our understanding of these differences offers to elucidate the molecular mechanisms underlying sex-specific traits and diseases. However, to date, most studies have either ignored the X Chromosome or had insufficient power to test for the sex-specific impact of genetic variation. By analyzing whole blood transcriptomes of 922 individuals, we have conducted the first large-scale, genome-wide analysis of the impact of both sex and genetic variation on patterns of gene expression, including comparison between the X Chromosome and autosomes. We identified a depletion of expression quantitative trait loci (eQTL) on the X Chromosome, especially among genes under high selective constraint. In contrast, we discovered an enrichment of sex-specific regulatory variants on the X Chromosome. To resolve the molecular mechanisms underlying such effects, we generated chromatin accessibility data through ATAC-sequencing to connect sex-specific chromatin accessibility to sex-specific patterns of expression and regulatory variation. As sex-specific regulatory variants discovered in our study can inform sex differences in heritable disease prevalence, we integrated our data with genome-wide association study data for multiple immune traits identifying several traits with significant sex biases in genetic susceptibilities. Together, our study provides genome-wide insight into how genetic variation, the X Chromosome, and sex shape human gene regulation and disease.
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Cromossomos Humanos X/genética , Transcriptoma , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Caracteres SexuaisRESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deposition of amyloid beta (Aß) and dysregulation of neurotrophic signaling, causing synaptic dysfunction, loss of memory, and cell death. The expression of p75 neurotrophin receptor is elevated in the brain of AD patients, suggesting its involvement in this disease. However, the exact mechanism of its action is not yet clear. Here, we show that p75 interacts with beta-site amyloid precursor protein cleaving enzyme-1 (BACE1), and this interaction is enhanced in the presence of Aß. Our results suggest that the colocalization of BACE1 and amyloid precursor protein (APP) is increased in the presence of both Aß and p75 in cortical neurons. In addition, the localization of APP and BACE1 in early endosomes is increased in the presence of Aß and p75. An increased phosphorylation of APP-Thr668 and BACE1-Ser498 by c-Jun N-terminal kinase (JNK) in the presence of Aß and p75 could be responsible for this localization. In conclusion, our study proposes a potential involvement in amyloidogenesis for p75, which may represent a future therapeutic target for AD. Cover Image for this Issue: doi. 10.1111/jnc.14163.
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Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Córtex Cerebral/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Camundongos Knockout , Cultura Primária de Células , Receptores de Fator de Crescimento Neural/genética , Transdução de SinaisRESUMO
Genomic imprinting is an important regulatory mechanism that silences one of the parental copies of a gene. To systematically characterize this phenomenon, we analyze tissue specificity of imprinting from allelic expression data in 1582 primary tissue samples from 178 individuals from the Genotype-Tissue Expression (GTEx) project. We characterize imprinting in 42 genes, including both novel and previously identified genes. Tissue specificity of imprinting is widespread, and gender-specific effects are revealed in a small number of genes in muscle with stronger imprinting in males. IGF2 shows maternal expression in the brain instead of the canonical paternal expression elsewhere. Imprinting appears to have only a subtle impact on tissue-specific expression levels, with genes lacking a systematic expression difference between tissues with imprinted and biallelic expression. In summary, our systematic characterization of imprinting in adult tissues highlights variation in imprinting between genes, individuals, and tissues.
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Impressão Genômica , Genômica , Adulto , Alelos , Análise por Conglomerados , Metilação de DNA , Bases de Dados de Ácidos Nucleicos , Feminino , Regulação da Expressão Gênica , Variação Genética , Genótipo , Humanos , Masculino , Especificidade de Órgãos/genética , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Fatores SexuaisRESUMO
PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions > 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.
Assuntos
Estudos de Associação Genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Variação Genética , Genoma Humano , Genômica , Análise de Sequência de DNA , Criança , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Ecocardiografia , Genômica/métodos , Humanos , Masculino , Fenótipo , Análise de Sequência de DNA/métodos , Deleção de SequênciaRESUMO
PURPOSE: To compare the volumetric changes in successfully treated clefts with secondary alveolar grafting using recombinant human bone morphogenic protein-2 (rhBMP-2) delivered in ß-tricalcium phosphate (ßTCP) scaffold versus autogenous grafts obtained from the iliac crest and mandibular symphysis. PATIENTS AND METHODS: We performed a retrospective cohort study of cone-beam computed tomography scans of 25 subjects with unilateral or bilateral clefts. Of the 25 patients, 7 received an iliac crest bone graft, 9 received a mandibular symphyseal bone graft, and 9 subjects received the rhBMP-2/ßTCP bone substitute. Volumetric rendering software was used to calculate the amount of new bone formation and residual bone defect present in the cleft area. The data were analyzed using Wilcoxon and Kruskal-Wallis tests and Pearson's correlation coefficient. RESULTS: The mean percentage of new bone formation for the iliac crest, symphysis, and rhBMP-2/ßTCP was 85.47, 80.56, and 81.22%, respectively (P = .0854). The initial cleft volume had a weak positive correlation with the percentage of new bone formation (r = 0.18), but the postoperative residual cleft volume had a strong negative correlation (r = 0.71). CONCLUSIONS: rhBMP2 delivered in a ßTCP scaffold in alveolar cleft patients can be a viable alternative to autogenous iliac crest and symphysis grafts, eliminating donor site morbidity.
Assuntos
Enxerto de Osso Alveolar/métodos , Proteínas Morfogenéticas Ósseas/uso terapêutico , Fosfatos de Cálcio/uso terapêutico , Fenda Labial/cirurgia , Fissura Palatina/cirurgia , Ílio/transplante , Mandíbula/transplante , Criança , Fenda Labial/diagnóstico por imagem , Fissura Palatina/diagnóstico por imagem , Tomografia Computadorizada de Feixe Cônico , Feminino , Humanos , Masculino , Interpretação de Imagem Radiográfica Assistida por Computador , Estudos Retrospectivos , Resultado do TratamentoRESUMO
At least 15% of the disease-causing mutations affect mRNA splicing. Many splicing mutations are missed in a clinical setting due to limitations of in silico prediction algorithms or their location in noncoding regions. Whole-transcriptome sequencing is a promising new tool to identify these mutations; however, it will be a challenge to obtain disease-relevant tissue for RNA. Here, we describe an individual with a sporadic atypical spinal muscular atrophy, in whom clinical DNA sequencing reported one pathogenic ASAH1 mutation (c.458A>G;p.Tyr153Cys). Transcriptome sequencing on patient leukocytes identified a highly significant and atypical ASAH1 isoform not explained by c.458A>G(p<10-16 ). Subsequent Sanger-sequencing identified the splice mutation responsible for the isoform (c.504A>C;p.Lys168Asn) and provided a molecular diagnosis of autosomal-recessive spinal muscular atrophy with progressive myoclonic epilepsy. Our findings demonstrate the utility of RNA sequencing from blood to identify splice-impacting disease mutations for nonhematological conditions, providing a diagnosis for these otherwise unsolved patients.
Assuntos
Ceramidase Ácida/genética , Atrofia Muscular Espinal/sangue , Epilepsias Mioclônicas Progressivas/sangue , Splicing de RNA/genética , Ceramidase Ácida/sangue , Pré-Escolar , Humanos , Masculino , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/genética , Mutação , Epilepsias Mioclônicas Progressivas/complicações , Epilepsias Mioclônicas Progressivas/genética , Patologia Molecular , Análise de Sequência de DNA , Transcriptoma/genéticaRESUMO
Recent and rapid human population growth has led to an excess of rare genetic variants that are expected to contribute to an individual's genetic burden of disease risk. To date, much of the focus has been on rare protein-coding variants, for which potential impact can be estimated from the genetic code, but determining the impact of rare noncoding variants has been more challenging. To improve our understanding of such variants, we combined high-quality genome sequencing and RNA sequencing data from a 17-individual, three-generation family to contrast expression quantitative trait loci (eQTLs) and splicing quantitative trait loci (sQTLs) within this family to eQTLs and sQTLs within a population sample. Using this design, we found that eQTLs and sQTLs with large effects in the family were enriched with rare regulatory and splicing variants (minor allele frequency < 0.01). They were also more likely to influence essential genes and genes involved in complex disease. In addition, we tested the capacity of diverse noncoding annotation to predict the impact of rare noncoding variants. We found that distance to the transcription start site, evolutionary constraint, and epigenetic annotation were considerably more informative for predicting the impact of rare variants than for predicting the impact of common variants. These results highlight that rare noncoding variants are important contributors to individual gene-expression profiles and further demonstrate a significant capability for genomic annotation to predict the impact of rare noncoding variants.
Assuntos
Genoma Humano , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas , RNA não Traduzido/genética , Análise de Sequência de RNA , Transcriptoma , Família , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfócitos/metabolismo , População Branca/genéticaRESUMO
We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , RNA/análise , Análise de Sequência de RNA/métodos , Alelos , Encéfalo/metabolismo , Encéfalo/patologia , Código de Barras de DNA Taxonômico , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Genótipo , Humanos , Edição de RNA , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de RNA/economia , TranscriptomaRESUMO
Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.
Assuntos
Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Células-Tronco Hematopoéticas , Histona-Lisina N-Metiltransferase/genética , Leucemia Aguda Bifenotípica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Antígenos CD34/imunologia , Separação Celular , Técnicas de Introdução de Genes , Genoma Humano , Humanos , Camundongos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução Genética , TransfecçãoRESUMO
Personal exome and genome sequencing provides access to loss-of-function and rare deleterious alleles whose interpretation is expected to provide insight into individual disease burden. However, for each allele, accurate interpretation of its effect will depend on both its penetrance and the trait's expressivity. In this regard, an important factor that can modify the effect of a pathogenic coding allele is its level of expression; a factor which itself characteristically changes across tissues. To better inform the degree to which pathogenic alleles can be modified by expression level across multiple tissues, we have conducted exome, RNA and deep, targeted allele-specific expression (ASE) sequencing in ten tissues obtained from a single individual. By combining such data, we report the impact of rare and common loss-of-function variants on allelic expression exposing stronger allelic bias for rare stop-gain variants and informing the extent to which rare deleterious coding alleles are consistently expressed across tissues. This study demonstrates the potential importance of transcriptome data to the interpretation of pathogenic protein-coding variants.
Assuntos
Alelos , Proteínas/genética , Exoma , Humanos , Reação em Cadeia da PolimeraseRESUMO
Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%-48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels.
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Evolução Molecular , Genoma Humano , Mutação INDEL/genética , Genética Populacional , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutagênese Insercional , Taxa de Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways, including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains the lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.
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Hematopoese , Proteínas de Homeodomínio/metabolismo , Células Progenitoras Linfoides/fisiologia , Células Progenitoras Mieloides/fisiologia , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula/genética , Sobrevivência Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteína Meis1 , Fator de Transcrição 1 de Leucemia de Células Pré-B , Multimerização Proteica , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.
Assuntos
Transformação Celular Neoplásica , Quinase 3 da Glicogênio Sintase/metabolismo , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/patologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Animais , Divisão Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27 , Modelos Animais de Doenças , Fase G1 , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/deficiência , Quinase 3 da Glicogênio Sintase/genética , Histona-Lisina N-Metiltransferase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Leucemia Linfoide/enzimologia , Leucemia Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Células Precursoras de Linfócitos B/enzimologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Proto-Oncogene MasRESUMO
Transcriptomics is a powerful tool for unraveling the molecular effects of genetic variants and disease diagnosis. Prior studies have demonstrated that choice of genome build impacts variant interpretation and diagnostic yield for genomic analyses. To identify the extent genome build also impacts transcriptomics analyses, we studied the effect of the hg19, hg38, and CHM13 genome builds on expression quantification and outlier detection in 386 rare disease and familial control samples from both the Undiagnosed Diseases Network (UDN) and Genomics Research to Elucidate the Genetics of Rare Disease (GREGoR) Consortium. We identified 2,800 genes with build-dependent quantification across six routinely-collected biospecimens, including 1,391 protein-coding genes and 341 known rare disease genes. We further observed multiple genes that only have detectable expression in a subset of genome builds. Finally, we characterized how genome build impacts the detection of outlier transcriptomic events. Combined, we provide a database of genes impacted by build choice, and recommend that transcriptomics-guided analyses and diagnoses are cross-referenced with these data for robustness.
RESUMO
Regular exercise has many physical and brain health benefits, yet the molecular mechanisms mediating exercise effects across tissues remain poorly understood. Here we analyzed 400 high-quality DNA methylation, ATAC-seq, and RNA-seq datasets from eight tissues from control and endurance exercise-trained (EET) rats. Integration of baseline datasets mapped the gene location dependence of epigenetic control features and identified differing regulatory landscapes in each tissue. The transcriptional responses to 8 weeks of EET showed little overlap across tissues and predominantly comprised tissue-type enriched genes. We identified sex differences in the transcriptomic and epigenomic changes induced by EET. However, the sex-biased gene responses were linked to shared signaling pathways. We found that many G protein-coupled receptor-encoding genes are regulated by EET, suggesting a role for these receptors in mediating the molecular adaptations to training across tissues. Our findings provide new insights into the mechanisms underlying EET-induced health benefits across organs.