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1.
Am J Hum Genet ; 108(8): 1436-1449, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216551

RESUMO

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.


Assuntos
Aberrações Cromossômicas , Análise Citogenética/métodos , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Genoma Humano , Mutação , Variações do Número de Cópias de DNA , Feminino , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Masculino , Análise de Sequência de DNA
2.
Nat Commun ; 11(1): 4912, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999275

RESUMO

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.


Assuntos
Glicemia/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Ilhotas Pancreáticas/metabolismo , Locos de Características Quantitativas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Glicemia/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Diabetes Mellitus Tipo 2/sangue , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA-Seq , Análise de Sequência de DNA , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Adulto Jovem
3.
PLoS Genet ; 16(9): e1009019, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32915782

RESUMO

Loci identified in genome-wide association studies (GWAS) can include multiple distinct association signals. We sought to identify the molecular basis of multiple association signals for adiponectin, a hormone involved in glucose regulation secreted almost exclusively from adipose tissue, identified in the Metabolic Syndrome in Men (METSIM) study. With GWAS data for 9,262 men, four loci were significantly associated with adiponectin: ADIPOQ, CDH13, IRS1, and PBRM1. We performed stepwise conditional analyses to identify distinct association signals, a subset of which are also nearly independent (lead variant pairwise r2<0.01). Two loci exhibited allelic heterogeneity, ADIPOQ and CDH13. Of seven association signals at the ADIPOQ locus, two signals colocalized with adipose tissue expression quantitative trait loci (eQTLs) for three transcripts: trait-increasing alleles at one signal were associated with increased ADIPOQ and LINC02043, while trait-increasing alleles at the other signal were associated with decreased ADIPOQ-AS1. In reporter assays, adiponectin-increasing alleles at two signals showed corresponding directions of effect on transcriptional activity. Putative mechanisms for the seven ADIPOQ signals include a missense variant (ADIPOQ G90S), a splice variant, a promoter variant, and four enhancer variants. Of two association signals at the CDH13 locus, the first signal consisted of promoter variants, including the lead adipose tissue eQTL variant for CDH13, while a second signal included a distal intron 1 enhancer variant that showed ~2-fold allelic differences in transcriptional reporter activity. Fine-mapping and experimental validation demonstrated that multiple, distinct association signals at these loci can influence multiple transcripts through multiple molecular mechanisms.


Assuntos
Adiponectina/genética , Adiponectina/metabolismo , Tecido Adiposo/metabolismo , Alelos , Caderinas/genética , Caderinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Frequência do Gene/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Síndrome Metabólica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Nature ; 582(7811): 240-245, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499647

RESUMO

Meta-analyses of genome-wide association studies (GWAS) have identified more than 240 loci that are associated with type 2 diabetes (T2D)1,2; however, most of these loci have been identified in analyses of individuals with European ancestry. Here, to examine T2D risk in East Asian individuals, we carried out a meta-analysis of GWAS data from 77,418 individuals with T2D and 356,122 healthy control individuals. In the main analysis, we identified 301 distinct association signals at 183 loci, and across T2D association models with and without consideration of body mass index and sex, we identified 61 loci that are newly implicated in predisposition to T2D. Common variants associated with T2D in both East Asian and European populations exhibited strongly correlated effect sizes. Previously undescribed associations include signals in or near GDAP1, PTF1A, SIX3, ALDH2, a microRNA cluster, and genes that affect the differentiation of muscle and adipose cells3. At another locus, expression quantitative trait loci at two overlapping T2D signals affect two genes-NKX6-3 and ANK1-in different tissues4-6. Association studies in diverse populations identify additional loci and elucidate disease-associated genes, biology, and pathways.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Aldeído-Desidrogenase Mitocondrial/genética , Alelos , Anquirinas/genética , Índice de Massa Corporal , Estudos de Casos e Controles , Europa (Continente)/etnologia , Proteínas do Olho/genética , Extremo Oriente/etnologia , Feminino , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/análise , Fatores de Transcrição/genética , Transcrição Genética
5.
Am J Hum Genet ; 106(1): 112-120, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31883642

RESUMO

Whole-genome sequencing (WGS) can improve assessment of low-frequency and rare variants, particularly in non-European populations that have been underrepresented in existing genomic studies. The genetic determinants of C-reactive protein (CRP), a biomarker of chronic inflammation, have been extensively studied, with existing genome-wide association studies (GWASs) conducted in >200,000 individuals of European ancestry. In order to discover novel loci associated with CRP levels, we examined a multi-ancestry population (n = 23,279) with WGS (∼38× coverage) from the Trans-Omics for Precision Medicine (TOPMed) program. We found evidence for eight distinct associations at the CRP locus, including two variants that have not been identified previously (rs11265259 and rs181704186), both of which are non-coding and more common in individuals of African ancestry (∼10% and ∼1% minor allele frequency, respectively, and rare or monomorphic in 1000 Genomes populations of East Asian, South Asian, and European ancestry). We show that the minor (G) allele of rs181704186 is associated with lower CRP levels and decreased transcriptional activity and protein binding in vitro, providing a plausible molecular mechanism for this African ancestry-specific signal. The individuals homozygous for rs181704186-G have a mean CRP level of 0.23 mg/L, in contrast to individuals heterozygous for rs181704186 with mean CRP of 2.97 mg/L and major allele homozygotes with mean CRP of 4.11 mg/L. This study demonstrates the utility of WGS in multi-ethnic populations to drive discovery of complex trait associations of large effect and to identify functional alleles in noncoding regulatory regions.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Grupo com Ancestrais do Continente Asiático/genética , Proteína C-Reativa/genética , Grupo com Ancestrais do Continente Europeu/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodos , Estudos de Coortes , Frequência do Gene , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação
6.
Hum Mol Genet ; 28(6): 888-895, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445632

RESUMO

Total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are heritable risk factors for cardiovascular disease, yet the molecular mechanisms underlying the majority of blood lipid-associated genome-wide association studies signals remain elusive. One association signal is located in intron 3 of VLDLR; rs3780181-A is a risk allele associated (P ≤ 2 × 10-9) with increased TC and LDL-C. We investigated variants, genes and mechanisms underlying this association signal. We used a functional genetic approach to show that the intronic region spanning rs3780181 exhibited 1.6-7.6-fold enhancer activity in human HepG2 hepatocyte, THP-1 monocyte and Simpson-Golabi-Behmel Syndrome (SGBS) preadipocyte cells and that the rs3780181-A risk allele showed significantly less enhancer activity compared with the G allele, consistent with the direction of an expression quantitative trait locus in liver. In addition, rs3780181 alleles showed differential binding to multiple nuclear proteins, including stronger IRF2 binding to the rs3780181 G allele. We used a CRISPR-cas9 approach to delete 475 and 663 bp of the putative enhancer element in HEK293T kidney cells; compared to expression of mock-edited cell lines, the homozygous enhancer deletion cell lines showed 1.2-fold significantly (P < 0.04) decreased expression of VLDLR, as well as 1.5-fold decreased expression of SMARCA2, located 388 kb away. Together, these results identify an enhancer of VLDLR expression and suggest that altered binding of one or more factors bound to rs3780181 alleles decreases enhancer activity and reduces at least VLDLR expression, leading to increased TC and LDL-C.


Assuntos
Alelos , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Locos de Características Quantitativas , Receptores de LDL/genética , Deleção de Sequência , Biologia Computacional/métodos , Sequência Conservada , Predisposição Genética para Doença , Variação Genética , Humanos , Fator Regulador 2 de Interferon/metabolismo , Anotação de Sequência Molecular , Motivos de Nucleotídeos , Polimorfismo de Nucleotídeo Único , Ligação Proteica
7.
Cereb Cortex ; 27(9): 4396-4410, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27578493

RESUMO

Cortical oscillations modulate cellular excitability and facilitate neuronal communication and information processing. Layer 5 pyramidal cells (L5 PYs) drive low-frequency oscillations (<4 Hz) in neocortical networks in vivo. In vitro, individual L5 PYs exhibit subthreshold resonance in the theta band (4-8 Hz). This bandpass filtering of periodic input is mediated by h-current (Ih) and m-current (IM) that selectively suppress low-frequency input. It has remained unclear how these intrinsic properties of cells contribute to the emergent, network oscillation dynamics. To begin to close this gap, we studied the link between cellular and network mechanisms of network resonance driven by L5 PYs. We performed multielectrode array recordings of network activity in slices of medial prefrontal cortex from the Thy1-ChR2-eYFP line and activated the network by temporally patterned optogenetic suprathreshold stimulation. Networks driven by stimulation of L5 PYs exhibited resonance in the theta band. We found that Ih and IM play a role in resonant suprathreshold network response to depolarizing stimuli. The action of Ih in mediating resonance was dependent on synaptic transmission while that of IM was not. These results demonstrate how synergistic interaction of synaptic and intrinsic ion channels contribute to the response of networks driven by L5 PYs.


Assuntos
Neurônios/fisiologia , Células Piramidais/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica/métodos , Hipocampo/fisiologia , Canais Iônicos/fisiologia , Camundongos , Rede Nervosa/fisiologia
8.
Brain Stimul ; 7(6): 878-89, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25129402

RESUMO

BACKGROUND: Transcranial alternating current stimulation (tACS) is a non-invasive brain stimulation modality that may modulate cognition by enhancing endogenous neocortical oscillations by application of sine-wave electric fields. Yet, the role of endogenous network activity in enabling and shaping the effects of tACS has remained unclear. OBJECTIVE: We combined optogenetic stimulation and multichannel slice electrophysiology to elucidate how the effect of a weak sine-wave electric field depends on the ongoing cortical oscillatory activity. We hypothesized that endogenous cortical oscillations constrain neuromodulation by tACS. METHODS: We studied the effect of weak sine-wave electric fields on oscillatory activity in mouse neocortical slices. Optogenetic control of the network activity enabled the generation of in vivo-like cortical oscillations for studying the temporal relationship between network activity and sine-wave electric field stimulation. RESULTS: Weak electric fields enhanced endogenous oscillations but failed to induce a frequency shift of the ongoing oscillation for stimulation frequencies that were not matched to the endogenous oscillation. This constraint on the effect of electric field stimulation imposed by endogenous network dynamics was limited to the case of weak electric fields targeting in vivo-like network dynamics. Together, these results suggest that the key mechanism of tACS may be enhancing, but not overriding, intrinsic network dynamics. CONCLUSION: Our results contribute to understanding the inconsistent tACS results from human studies and propose that stimulation precisely adjusted in frequency to the endogenous oscillations is key to rational design of non-invasive brain stimulation paradigms.


Assuntos
Neocórtex/fisiologia , Rede Nervosa/fisiologia , Estimulação Transcraniana por Corrente Contínua , Animais , Relógios Biológicos , Ondas Encefálicas/fisiologia , Camundongos , Optogenética , Células Piramidais/fisiologia
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