RESUMEN
Leprosy is a chronic infectious disease of the skin and peripheral nerves with a strong genetic predisposition. Recent genome-wide approaches have identified numerous common variants associated with leprosy, almost all in the Chinese population. We conducted the first family-based genome-wide association study of leprosy in 622 affected offspring from Vietnam, followed by replication in an independent sample of 1181 leprosy cases and 668 controls of the same ethnic origin. The most significant results were observed within the HLA region, in which six SNPs displayed genome-wide significant associations, all of which were replicated in the independent case/control sample. We investigated the signal in the HLA region in more detail, by conducting a multivariate analysis on the case/control sample of 319 GWAS-suggestive HLA hits for which evidence for replication was obtained. We identified three independently associated SNPs, two located in the HLA class I region (rs1265048: OR = 0.69 [0.58-0.80], combined p-value = 5.53x10-11; and rs114598080: OR = 1.47 [1.46-1.48], combined p-value = 8.77x10-13), and one located in the HLA class II region (rs3187964 (OR = 1.67 [1.55-1.80], combined p-value = 8.35x10-16). We also validated two previously identified risk factors for leprosy: the missense variant rs3764147 in the LACC1 gene (OR = 1.52 [1.41-1.63], combined p-value = 5.06x10-14), and the intergenic variant rs6871626 located close to the IL12B gene (OR = 0.73 [0.61-0.84], combined p-value = 6.44x10-8). These results shed new light on the genetic control of leprosy, by dissecting the influence of HLA SNPs, and validating the independent role of two additional variants in a large Vietnamese sample.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Lepra/genética , Polimorfismo de Nucleótido Simple , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Subunidad p40 de la Interleucina-12/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Lepra/epidemiología , MasculinoRESUMEN
PURPOSE: Treacher Collins syndrome (TCS) is a rare autosomal dominant mandibulofacial dysostosis, with a prevalence of 0.2-1/10,000. Features include bilateral and symmetrical malar and mandibular hypoplasia and facial abnormalities due to abnormal neural crest cell (NCC) migration and differentiation. To date, three genes have been identified: TCOF1, POLR1C, and POLR1D. Despite a large number of patients with a molecular diagnosis, some remain without a known genetic anomaly. METHODS: We performed exome sequencing for four individuals with TCS but who were negative for pathogenic variants in the known causative genes. The effect of the pathogenic variants was investigated in zebrafish. RESULTS: We identified three novel pathogenic variants in POLR1B. Knockdown of polr1b in zebrafish induced an abnormal craniofacial phenotype mimicking TCS that was associated with altered ribosomal gene expression, massive p53-associated cellular apoptosis in the neuroepithelium, and reduced number of NCC derivatives. CONCLUSION: Pathogenic variants in the RNA polymerase I subunit POLR1B might induce massive p53-dependent apoptosis in a restricted neuroepithelium area, altering NCC migration and causing cranioskeletal malformations. We identify POLR1B as a new causative gene responsible for a novel TCS syndrome (TCS4) and establish a novel experimental model in zebrafish to study POLR1B-related TCS.
Asunto(s)
Anomalías Craneofaciales/genética , ARN Polimerasas Dirigidas por ADN/genética , Disostosis Mandibulofacial/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Animales , Apoptosis/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Anomalías Craneofaciales/patología , Predisposición Genética a la Enfermedad , Humanos , Disostosis Mandibulofacial/patología , Mutación , Cresta Neural/anomalías , Cresta Neural/patología , Proteína p53 Supresora de Tumor/genética , Secuenciación del Exoma , Pez Cebra/genéticaRESUMEN
Pathogenic variants in BRCA1 and BRCA2 only explain the underlying genetic cause of about 10% of hereditary breast and ovarian cancer families. Because of cost-effectiveness, multigene panel testing is often performed even if the clinical utility of testing most of the genes remains questionable. The purpose of our study was to assess the contribution of rare, deleterious-predicted variants in DNA repair genes in familial breast cancer (BC) in a well-characterized and homogeneous population. We analyzed 113 DNA repair genes selected from either an exome sequencing or a candidate gene approach in the GENESIS study, which includes familial BC cases with no BRCA1 or BRCA2 mutation and having a sister with BC (N = 1,207), and general population controls (N = 1,199). Sequencing data were filtered for rare loss-of-function variants (LoF) and likely deleterious missense variants (MV). We confirmed associations between LoF and MV in PALB2, ATM and CHEK2 and BC occurrence. We also identified for the first time associations between FANCI, MAST1, POLH and RTEL1 and BC susceptibility. Unlike other associated genes, carriers of an ATM LoF had a significantly higher risk of developing BC than carriers of an ATM MV (ORLoF = 17.4 vs. ORMV = 1.6; p Het = 0.002). Hence, our approach allowed us to specify BC relative risks associated with deleterious-predicted variants in PALB2, ATM and CHEK2 and to add MAST1, POLH, RTEL1 and FANCI to the list of DNA repair genes possibly involved in BC susceptibility. We also highlight that different types of variants within the same gene can lead to different risk estimates.
Asunto(s)
Neoplasias de la Mama/genética , Reparación del ADN/genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/diagnóstico , Estudios de Casos y Controles , Femenino , Humanos , Persona de Mediana Edad , Medición de Riesgo/métodos , HermanosRESUMEN
The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting ß-cells and neurons and underline the existence of transnosology pathways in diabetes and its co-morbidities.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Autofagia , Sitios de Unión , Línea Celular Tumoral , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Hipocampo/citología , Masculino , Neurogénesis , Neuronas/citología , Células PC12 , Polimorfismo Genético , Unión Proteica , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Aristolochic acid (AA) is a nephrotoxicant associated with AA nephropathy (AAN) and upper urothelial tract cancer (UUTC). Whole-genome sequences of 14 Romanian cases of renal cell carcinoma (RCC) recently exhibited mutational signatures consistent with AA exposure, although RCC had not been previously linked with AAN and AA exposure was previously reported only in localised rural areas. METHODS: We performed mass spectrometric measurements of the aristolactam (AL) DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) in nontumour renal tissues of the 14 Romanian RCC cases and 15 cases from 3 other countries. RESULTS: We detected dA-AL-I in the 14 Romanian cases at levels ranging from 0.7 to 27 adducts per 10(8) DNA bases, in line with levels reported in Asian and Balkan populations exposed through herbal remedies or food contamination. The 15 cases from other countries were negative. INTERPRETATION: Although the source of exposure is uncertain and likely different in AAN regions than elsewhere, our results demonstrate that AA exposure in Romania exists outside localised AAN regions and provide further evidence implicating AA in RCC.
Asunto(s)
Ácidos Aristolóquicos/toxicidad , Carcinoma de Células Renales/inducido químicamente , Neoplasias Renales/inducido químicamente , Ácidos Aristolóquicos/análisis , Carcinoma de Células Renales/genética , Desoxiadenosinas/análisis , Humanos , Neoplasias Renales/genética , Mutación , Rumanía , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Characterizing the relationships between genomic and phenotypic variation is essential to understanding disease etiology. Information-dense data sets derived from pathophysiological, proteomic and transcriptomic profiling have been applied to map quantitative trait loci (QTLs). Metabolic traits, already used in QTL studies in plants, are essential phenotypes in mammalian genetics to define disease biomarkers. Using a complex mammalian system, here we show chromosomal mapping of untargeted plasma metabolic fingerprints derived from NMR spectroscopic analysis in a cross between diabetic and control rats. We propose candidate metabolites for the most significant QTLs. Metabolite profiling in congenic strains provided evidence of QTL replication. Linkage to a gut microbial metabolite (benzoate) can be explained by deletion of a uridine diphosphate glucuronosyltransferase. Mapping metabotypic QTLs provides a practical approach to understanding genome-phenotype relationships in mammals and may uncover deeper biological complexity, as extended genome (microbiome) perturbations that affect disease processes through transgenomic effects may influence QTL detection.
Asunto(s)
Diabetes Mellitus/genética , Ligamiento Genético , Genoma/genética , Metabolismo/genética , Fenotipo , Sitios de Carácter Cuantitativo , Animales , Secuencia de Bases , Benzoatos/química , Biomarcadores/análisis , Glucuronosiltransferasa/genética , Escala de Lod , Datos de Secuencia Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Ratas , Análisis de Secuencia de ADNRESUMEN
The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.
Asunto(s)
Proteínas Portadoras/genética , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Riñón/metabolismo , Riñón/patología , Proteínas Nucleares/genética , Animales , Repetición de Anquirina , Proteínas Portadoras/metabolismo , Cilios/metabolismo , Femenino , Homocigoto , Humanos , Riñón/embriología , Enfermedades Renales Quísticas/fisiopatología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/patología , Asa de la Nefrona/metabolismo , Asa de la Nefrona/patología , Masculino , Ratones , Ratones Endogámicos C3H , Mutación Missense , Proteínas Nucleares/metabolismo , Fenotipo , Podocitos/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Proteínas de Unión al ARN/metabolismo , RatasRESUMEN
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. METHODS: Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. RESULTS: Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. CONCLUSIONS: Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.
Asunto(s)
Benzofenonas/farmacología , Inhibidores de Catecol O-Metiltransferasa , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Nitrofenoles/farmacología , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedades Renales Poliquísticas/patología , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TolcaponaRESUMEN
With successes of genome-wide association studies, molecular phenotyping systems are developed to identify genetically determined disease-associated biomarkers. Genetic studies of the human metabolome are emerging but exclusively apply targeted approaches, which restricts the analysis to a limited number of well-known metabolites. We have developed novel technical and statistical methods for systematic and automated quantification of untargeted NMR spectral data designed to perform robust and accurate quantitative trait locus (QTL) mapping of known and previously unreported molecular compounds of the metabolome. For each spectral peak, six summary statistics were calculated and independently tested for evidence of genetic linkage in a cohort of F2 (129S6xBALB/c) mice. The most significant evidence of linkages were obtained with NMR signals characterizing the glycerate (LOD10-42) at the mutant glycerate kinase locus, which demonstrate the power of metabolomics in quantitative genetics to identify the biological function of genetic variants. These results provide new insights into the resolution of the complex nature of metabolic regulations and novel analytical techniques that maximize the full utilization of metabolomic spectra in human genetics to discover mappable disease-associated biomarkers.
Asunto(s)
Mapeo Cromosómico/métodos , Genómica/métodos , Ácidos Glicéricos/orina , Metaboloma/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Sitios de Carácter Cuantitativo , Análisis de Varianza , Animales , Simulación por Computador , Escala de Lod , Masculino , Metabolómica , Ratones , Ratones Endogámicos BALB C , Resonancia Magnética Nuclear Biomolecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Maintaining homeostasis in higher organisms involves a complex interplay of multiple ubiquitous and organ-specific molecular mechanisms that can be characterized using functional genomics technologies such as transcriptomics, proteomics, and metabonomics and dissected out through genetic investigations in healthy and diseased individuals. We characterized the genomic, metabolic, and physiological divergence of several inbred rat strains--Brown Norway, Lewis, Wistar Kyoto, Fisher (F344)--frequently used as healthy controls in genetic studies of the cardiometabolic syndrome. Hierarchical clustering of (1)H NMR-based metabolic profiles (n = 20 for urine, n = 16 for plasma) identified metabolic phenotype (metabotype) divergence patterns similar to the phylogenetic variability based on single nucleotide polymorphisms. However, the observed urinary metabotype variation exceeded that explainable by genetic polymorphisms. To understand further this natural variation, we used an integrative, knowledge-based network biology metabolic pathway analysis approach, coined Metabolite-Set Enrichment Analysis (MSEA). MSEA reveals that homeostasis and physiological plasticity can be achieved despite widespread divergences in glucose, lipid, amino acid, and energy metabolism in the host, together with different gut microbiota contributions suggestive of strain-specific transgenomic interactions. This work illustrates the concept of natural metabolomic variation, leading to physiologically stable albeit diverse strategies within the range of normality, all of which are highly relevant to animal model physiology, genetical genomics, and patient stratification in personalized healthcare.
Asunto(s)
Redes y Vías Metabólicas/fisiología , Metaboloma , Metabolómica/métodos , Ratas/metabolismo , Ratas/fisiología , Animales , Análisis por Conglomerados , Humanos , Masculino , Resonancia Magnética Nuclear Biomolecular , Fenotipo , Ratas EndogámicasRESUMEN
The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6((p.R823W))) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6((p.R823W)) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6((p.R823W)) cDNA in the renal tubular epithelium. The transgenic Anks6((p.R823W)) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6((p.R823W)) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.
Asunto(s)
Proteínas Nucleares/genética , Enfermedades Renales Poliquísticas/genética , Sustitución de Aminoácidos/genética , Animales , Arginina/genética , Expresión Génica/fisiología , Predisposición Genética a la Enfermedad , Masculino , Proteínas Mutantes/genética , Enfermedades Renales Poliquísticas/patología , Polimorfismo de Nucleótido Simple/fisiología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Triptófano/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Chronic obstructive pulmonary disease (COPD) is a destructive inflammatory disease and the genes expressed within the lung are crucial to its pathophysiology. We have determined the RNAseq transcriptome of bronchial brush cells from 312 stringently defined ex-smoker patients. Compared to healthy controls there were for males 40 differentially expressed genes (DEGs) and 73 DEGs for females with only 26 genes shared. The gene ontology (GO) term "response to bacterium" was shared, with several different DEGs contributing in males and females. Strongly upregulated genes TCN1 and CYP1B1 were unique to males and females, respectively. For male emphysema (E)-dominant and airway disease (A)-dominant COPD (defined by computed tomography) the term "response to stress" was found for both sub-phenotypes, but this included distinct up-regulated genes for the E-sub-phenotype (neutrophil-related CSF3R, CXCL1, MNDA) and for the A-sub-phenotype (macrophage-related KLF4, F3, CD36). In E-dominant disease, a cluster of mitochondria-encoded (MT) genes forms a signature, able to identify patients with emphysema features in a confirmation cohort. The MT-CO2 gene is upregulated transcriptionally in bronchial epithelial cells with the copy number essentially unchanged. Both MT-CO2 and the neutrophil chemoattractant CXCL1 are induced by reactive oxygen in bronchial epithelial cells. Of the female DEGs unique for E- and A-dominant COPD, 88% were detected in females only. In E-dominant disease we found a pronounced expression of mast cell-associated DEGs TPSB2, TPSAB1 and CPA3. The differential genes discovered in this study point towards involvement of different types of leukocytes in the E- and A-dominant COPD sub-phenotypes in males and females.
Asunto(s)
Susceptibilidad a Enfermedades , Expresión Génica , Leucocitos/metabolismo , Mitocondrias/genética , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Mucosa Respiratoria/metabolismo , Biomarcadores , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Leucocitos/inmunología , Leucocitos/patología , Masculino , Mitocondrias/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Factores Sexuales , TranscriptomaRESUMEN
BACKGROUND: Microarray technologies are widely used to quantify the abundance of transcripts corresponding to thousands of genes. To maximise the robustness of transcriptome results, we have tested the performance and reproducibility of rat and mouse gene expression data obtained with Affymetrix, Illumina and Operon platforms. RESULTS: We present a thorough analysis of the degree of reproducibility provided by analysing the transcriptomic profile of the same animals of several experimental groups under different popular microarray technologies in different tissues. Concordant results from inter- and intra-platform comparisons were maximised by testing many popular computational methods for generating fold changes and significances and by only considering oligonucleotides giving high expression levels. The choice of Affymetrix signal extraction technique was shown to have the greatest effect on the concordance across platforms. In both species, when choosing optimal methods, the agreement between data generated on the Affymetrix and Illumina was excellent; this was verified using qRT-PCR on a selection of genes present on all platforms. CONCLUSION: This study provides an extensive assessment of analytical methods best suited for processing data from different microarray technologies and can assist integration of technologically different gene expression datasets in biological systems.
Asunto(s)
Diabetes Mellitus Experimental/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas BN , Ratas Endogámicas WKY , Reproducibilidad de los ResultadosRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans. Although disease-causing mutations have been found in two genes, PKD1 and PKD2, a small number of ADPKD families exist that are unlinked to either of these genes, suggesting involvement of a third, as yet unidentified PKD3 gene. Susceptibility to renal cyst formation in the (cy/+) rat is caused by a missense mutation in Pkdr1 encoding the novel protein SamCystin. To initiate studies of the human orthologous gene, we determined the location and the organization of human PKDR1. We genotyped microsatellite markers flanking the human ortholog in PKD families that either are unlinked to known PKD genes, or in which mutations have not yet been identified and carried out mutation analysis in PKD patients. We identified eight novel single nucleotide polymorphisms, including three leading to amino acid changes. These variants are unlikely to account for PKD in these patients, yet the screening of other affected populations may provide information about the involvement of PKDR1 as a modifier gene in cystic kidney disease.
Asunto(s)
Sustitución de Aminoácidos/genética , Genoma Humano , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Mutación Puntual , Riñón Poliquístico Autosómico Dominante/genética , Canales Catiónicos TRPP/química , Canales Catiónicos TRPP/metabolismo , Animales , Humanos , Mutación Missense , RatasRESUMEN
The inbred Goto-Kakizaki (GK) rat strain is a unique model of spontaneous type 2 diabetes mellitus caused by naturally occurring genetic variants that have been selectively isolated from an outbred colony of Wistar rats. Genetic and genomic studies in experimental crosses and congenic strains of the GK have shed light on the complex etiopathogenesis of diabetes phenotypes in this model. Diabetes-related phenotypes in the GK are under polygenic control and distinct genetic loci regulate glucose tolerance, insulin secretion, ß-cell mass and plasma lipids. Metabolome and transcriptome profiling data in GK crosses and congenics, combined with GK genome resequencing, have resulted in a comprehensive landscape of genomic regulations of metabolism that can disentangle causal relationships between GK variants and diabetes phenotypes. Application of systems biology and systems genetics in the GK has contributed to improve our understanding of the fundamental mechanisms regulating metabolism. The wealth of physiological, genetic and genomic information in this strain makes it one of the most powerful model systems to improve our understanding of genetic regulations of metabolism and for testing therapeutic solutions for diabetes.
Asunto(s)
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/etiología , Regulación de la Expresión Génica , Animales , Mapeo Cromosómico , Diabetes Mellitus Tipo 2/genética , Fenotipo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Ratas Endogámicas , Ratas WistarRESUMEN
To test the impact of genetic heterogeneity on cis- and trans-mediated mechanisms of gene expression regulation, we profiled the transcriptome of adipose tissue in 20 inbred congenic strains derived from diabetic Goto-Kakizaki (GK) rats and Brown-Norway (BN) controls, which contain well-defined blocks (1-183 Mb) of genetic polymorphisms, and in 123 genetically heterogeneous rats of an (GK × BN)F2 offspring. Within each congenic we identified 73-1351 differentially expressed genes (DEGs), only 7.7% of which mapped within the congenic blocks, and which may be regulated in cis The remainder localized outside the blocks, and therefore must be regulated in trans Most trans-regulated genes exhibited approximately twofold expression changes, consistent with monoallelic expression. Altered biological pathways were replicated between congenic strains sharing blocks of genetic polymorphisms, but polymorphisms at different loci also had redundant effects on transcription of common distant genes and pathways. We mapped 2735 expression quantitative trait loci (eQTL) in the F2 cross, including 26% predominantly cis-regulated genes, which validated DEGs in congenic strains. A hotspot of >300 eQTL in a 10 cM region of chromosome 1 was enriched in DEGs in a congenic strain. However, many DEGs among GK, BN and congenic strains did not replicate as eQTL in F2 hybrids, demonstrating distinct mechanisms of gene expression when alleles segregate in an outbred population or are fixed homozygous across the entire genome or in short genomic regions. Our analysis provides conceptual advances in our understanding of the complex architecture of genome expression and pathway regulation, and suggests a prominent impact of epistasis and monoallelic expression on gene transcription.
RESUMEN
Spontaneous type 1 diabetes in BB rats is dependent on the RT1(u) MHC haplotype and homozygosity for an allele at the Lyp locus, which is responsible for a peripheral T-lymphopenia. Genetic studies have shown that there are other, as yet unidentified, genetic loci contributing to diabetes susceptibility in this strain. BB rats carrying wild-type Lyp alleles are not lymphopenic and are resistant to spontaneous diabetes (DR). Here we show that thymectomy and exposure to one sublethal dose of gamma-irradiation (TX-R) at 4 weeks of age result in the rapid development of insulitis followed by diabetes in 100% of DR rats. Administration of CD4(+)45RC(-) T-cells from unmanipulated, syngeneic donors immediately after irradiation prevents the disease. Splenic T-cells from TX-R-induced diabetic animals adoptively transfer type 1 diabetes to T-deficient recipients. ACI, WF, WAG, BN, LEW, PVG, and PVG.RT1(u) strains are resistant to TX-R-induced insulitis/diabetes. Genetic analyses revealed linkage between regions on chromosomes 1, 3, 4, 6, 9, and 16, and TX-R-induced type 1 diabetes in a cohort of nonlymphopenic F(2) (Wistar Furth x BBDP) animals. This novel model of TX-R-induced diabetes in nonlymphopenic BB rats can be used to identify environmental and cellular factors that are responsible for the initiation of antipancreatic autoimmunity.
Asunto(s)
Diabetes Mellitus Tipo 1 , Linfopenia/genética , Timectomía , Traslado Adoptivo , Factores de Edad , Animales , Autoinmunidad/genética , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/cirugía , Ligamiento Genético , Haplotipos , Antígenos Comunes de Leucocito/inmunología , Linfopenia/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/inmunología , Ratas , Ratas Endogámicas BB , Ratas Endogámicas WF , Especificidad de la Especie , Linfocitos T/inmunologíaRESUMEN
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.
Asunto(s)
Repetición de Anquirina/genética , Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Cilios/metabolismo , Riñón/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Vasopresinas/metabolismo , Secuencias de Aminoácidos/genética , Animales , Apoptosis/genética , Proteínas Portadoras/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Cilios/genética , Regulación hacia Abajo/genética , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Unión Proteica/genética , Transducción de Señal/genética , Vasopresinas/genéticaRESUMEN
Genetic studies in human populations and rodent models have identified regions of human chromosome 1q21-25 and rat chromosome 2 showing evidence of significant and replicated linkage to diabetes-related phenotypes. To investigate the relationship between the human and rat diabetes loci, we fine mapped the rat locus Nidd/gk2 linked to hyperinsulinemia in an F2 cross derived from the diabetic (type 2) Goto-Kakizaki (GK) rat and the Brown Norway (BN) control rat, and carried out its genetic and pathophysiological characterization in BN.GK congenic strains. Evidence of glucose intolerance and enhanced insulin secretion in a congenic strain allowed us to localize the underlying diabetes gene(s) in a rat chromosomal interval of approximately 3-6 cM conserved with an 11-Mb region of human 1q21-23. Positional diabetes candidate genes were tested for transcriptional changes between congenics and controls and sequence variations in a panel of inbred rat strains. Congenic strains of the GK rats represent powerful novel models for accurately defining the pathophysiological impact of diabetes gene(s) at the locus Nidd/gk2 and improving functional annotations of diabetes candidates in human 1q21-23.
Asunto(s)
Cromosomas Humanos Par 1/genética , Secuencia Conservada/genética , Diabetes Mellitus Tipo 2/genética , Sitios de Carácter Cuantitativo/genética , Animales , Animales Congénicos , Peso Corporal , Cruzamientos Genéticos , Femenino , Perfilación de la Expresión Génica , Genómica , Glucosa/farmacología , Intolerancia a la Glucosa/genética , Humanos , Hiperinsulinismo/genética , Insulina/metabolismo , Secreción de Insulina , Lípidos/sangre , Masculino , Fenotipo , Polimorfismo Genético/genética , Ratas , Ratas Endogámicas BN , Ratas Endogámicas , Análisis de Secuencia de ADN , Transcripción Genética/genéticaRESUMEN
Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.