RESUMEN
BACKGROUND: The genetic regulation of metabolic phenotypes (i.e., metabotypes) in type 2 diabetes mellitus occurs through complex organ-specific cellular mechanisms and networks contributing to impaired insulin secretion and insulin resistance. Genome-wide gene expression profiling systems can dissect the genetic contributions to metabolome and transcriptome regulations. The integrative analysis of multiple gene expression traits and metabolic phenotypes (i.e., metabotypes) together with their underlying genetic regulation remains a challenge. Here, we introduce a systems genetics approach based on the topological analysis of a combined molecular network made of genes and metabolites identified through expression and metabotype quantitative trait locus mapping (i.e., eQTL and mQTL) to prioritise biological characterisation of candidate genes and traits. METHODS: We used systematic metabotyping by 1H NMR spectroscopy and genome-wide gene expression in white adipose tissue to map molecular phenotypes to genomic blocks associated with obesity and insulin secretion in a series of rat congenic strains derived from spontaneously diabetic Goto-Kakizaki (GK) and normoglycemic Brown-Norway (BN) rats. We implemented a network biology strategy approach to visualize the shortest paths between metabolites and genes significantly associated with each genomic block. RESULTS: Despite strong genomic similarities (95-99 %) among congenics, each strain exhibited specific patterns of gene expression and metabotypes, reflecting the metabolic consequences of series of linked genetic polymorphisms in the congenic intervals. We subsequently used the congenic panel to map quantitative trait loci underlying specific mQTLs and genome-wide eQTLs. Variation in key metabolites like glucose, succinate, lactate, or 3-hydroxybutyrate and second messenger precursors like inositol was associated with several independent genomic intervals, indicating functional redundancy in these regions. To navigate through the complexity of these association networks we mapped candidate genes and metabolites onto metabolic pathways and implemented a shortest path strategy to highlight potential mechanistic links between metabolites and transcripts at colocalized mQTLs and eQTLs. Minimizing the shortest path length drove prioritization of biological validations by gene silencing. CONCLUSIONS: These results underline the importance of network-based integration of multilevel systems genetics datasets to improve understanding of the genetic architecture of metabotype and transcriptomic regulation and to characterize novel functional roles for genes determining tissue-specific metabolism.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Metaboloma , Sitios de Carácter Cuantitativo , Carácter Cuantitativo Heredable , Transcriptoma , Animales , Animales Congénicos , Mapeo Cromosómico , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Redes y Vías Metabólicas , Anotación de Secuencia Molecular , Ratas Endogámicas BN , Biología de SistemasRESUMEN
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
The R620W variant of PTPN22 is one of the major genetic risk factors for several autoimmune disorders including type 1 diabetes (T1D) in humans. In the BioBreeding T1D-prone (BBDP) rat, a single nucleotide polymorphism in Ptpn22 results in an A629T substitution immediately C-terminal to the aliphatic residues central to the Ptpn22-C-terminal Src kinase interaction. This variant exhibits a 50% decrease in C-terminal Src kinase binding affinity and contributes to T cell hyperresponsiveness. Examination of BBDP sublines congenic for the Iddm26.2 locus that includes Ptpn22 has not only shown an expansion of activated CD4(+)25(+) T lymphocytes in animals homozygous for the BBDP allele, consistent with enhanced TCR-mediated signaling, but also a decrease in their proportion of peripheral Foxp3(+) regulatory T cells. Furthermore, clinical assessment of both an F2(BBDP × ACI.1u.Lyp) cohort and Iddm26.2 congenic BBDP sublines has revealed an association of Ptpn22 with T1D. Specifically, in both cases, T1D risk is significantly greater in BBDP Ptpn22 homozygous and heterozygous animals. These findings are consistent with a role for rat Ptpn22 allelic variation within Iddm26.2 in the regulation of T cell responses, and subsequently the risk for development of T1D.
Asunto(s)
Alelos , Diabetes Mellitus Tipo 1/genética , Mutación Missense , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Sustitución de Aminoácidos , Animales , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Ratas , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
The autoimmune diabetic syndrome of the BioBreeding diabetes-prone (BBDP) rat is a polygenic disease that resembles in many aspects human type 1 diabetes (T1D). A successful approach to gain insight into the mechanisms underlying genetic associations in autoimmune diseases has been to identify and map disease-related subphenotypes that are under simpler genetic control than the full-blown disease. In this study, we focused on the ß cell overexpression of Ccl11 (Eotaxin), previously postulated to be diabetogenic in BBDR rats, a BBDP-related strain. We tested the hypothesis that this trait is genetically determined and contributes to the regulation of diabetes in BBDP rats. Similar to the BBDR strain, we observed a time-dependent, insulitis-independent pancreatic upregulation of Ccl11 in BBDP rats when compared with T1D-resistant ACI.1u.lyp animals. Through linkage analysis of a cross-intercross of these two parental strains, this trait was mapped to a region on chromosome 12 that overlaps Iddm30. Linkage results were confirmed by phenotypic assessment of a novel inbred BBDP.ACI-Iddm30 congenic line. As expected, the Iddm30 BBDP allele is associated with a significantly higher pancreatic expression of Ccl11; however, the same allele confers resistance to T1D. Analysis of islet-infiltrating T cells in Iddm30 congenic BBDP animals revealed that overexpression of pancreatic Ccl11, a prototypical Th2 chemokine, is associated with an enrichment in Th2 CD4+ T cells within the insulitic lesions. These results indicate that, in the BBDP rat, Iddm30 controls T1D susceptibility through both the regulation of Ccl11 expression in ß cells and the subsequent Th1/Th2 balance within islet-infiltrating T lymphocytes.
Asunto(s)
Quimiocina CCL11/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Regulación de la Expresión Génica , Sitios Genéticos/genética , Páncreas/inmunología , Páncreas/metabolismo , Balance Th1 - Th2 , Animales , Cruzamiento , Epistasis Genética , Femenino , Expresión Génica , Ligamiento Genético , Genotipo , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Fenotipo , Ratas , Ratas Endogámicas BB , Transcripción GenéticaRESUMEN
OBJECTIVE: Two type 1 diabetes susceptibility genes have been identified in the spontaneously diabetic biobreeding diabetes-prone (BBDP) rat, the major histocompatibility complex (MHC) (RT1) class II u haplotype (Iddm1) and Gimap5 (Iddm2). The strong effects of these have impeded previous efforts to map additional loci. We tested the hypothesis that type 1 diabetes is a polygenic disease in the BBDP rat. RESEARCH DESIGN AND METHODS: We performed the most comprehensive genome-wide linkage analysis for type 1 diabetes, age of disease onset (AOO), and insulitis subphenotypes in 574 F2 animals from a cross-intercross between BBDP and type 1 diabetes-resistant, double congenic ACI.BBDP-RT1u,Gimap5 (ACI.BB(1u.lyp)) rats, where both Iddm1 and Iddm2 were fixed as BBDP. RESULTS: A total of 19% of these F2 animals developed type 1 diabetes, and eight type 1 diabetes susceptibility loci were mapped, six showing significant linkage (chromosomes 1, 3, 6 [two loci], 12, and 14) and two (chromosomes 2 and 17) suggestive linkage. The chromosomes 6, 12, and 14 intervals were also linked to the severity of islet infiltration by immunocytes, while those on chromosomes 1, 6 (two loci), 14, 17, and a type 1 diabetes-unlinked chromosome 8 interval showed significant linkage to the degree of islet atrophy. Four loci exhibited suggestive linkage to AOO on chromosomes 2 (two loci), 7, and 18 but were unlinked to type 1 diabetes. INS, PTPN22, IL2/IL21, C1QTNF6, and C12orf30, associated with human type 1 diabetes, are contained within the chromosomes 1, 2, 7, and 12 loci. CONCLUSIONS: This study demonstrates that the BBDP diabetic syndrome is a complex, polygenic disease that may share additional susceptibility genes besides MHC class II with human type 1 diabetes.
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Diabetes Mellitus Tipo 1/genética , Ratas Endogámicas BB/genética , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Genoma , Glucosuria , Humanos , Modelos Genéticos , RatasRESUMEN
BACKGROUND: Complex etiology and pathogenesis of pathophysiological components of the cardio-metabolic syndrome have been demonstrated in humans and animal models. METHODOLOGY/PRINCIPAL FINDINGS: We have generated extensive physiological, genetic and genome-wide gene expression profiles in a congenic strain of the spontaneously diabetic Goto-Kakizaki (GK) rat containing a large region (110 cM, 170 Mb) of rat chromosome 1 (RNO1), which covers diabetes and obesity quantitative trait loci (QTL), introgressed onto the genetic background of the normoglycaemic Brown Norway (BN) strain. This novel disease model, which by the length of the congenic region closely mirrors the situation of a chromosome substitution strain, exhibits a wide range of abnormalities directly relevant to components of the cardio-metabolic syndrome and diabetes complications, including hyperglycaemia, hyperinsulinaemia, enhanced insulin secretion both in vivo and in vitro, insulin resistance, hypertriglyceridemia and altered pancreatic and renal histological structures. Gene transcription data in kidney, liver, skeletal muscle and white adipose tissue indicate that a disproportionately high number (43-83%) of genes differentially expressed between congenic and BN rats map to the GK genomic interval targeted in the congenic strain, which represents less than 5% of the total length of the rat genome. Genotype analysis of single nucleotide polymorphisms (SNPs) in strains genetically related to the GK highlights clusters of conserved and strain-specific variants in RNO1 that can assist the identification of naturally occurring variants isolated in diabetic and hypertensive strains when different phenotype selection procedures were applied. CONCLUSIONS: Our results emphasize the importance of rat congenic models for defining the impact of genetic variants in well-characterised QTL regions on in vivo pathophysiological features and cis-/trans- regulation of gene expression. The congenic strain reported here provides a novel and sustainable model for investigating the pathogenesis and genetic basis of risks factors for the cardio-metabolic syndrome.
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Diabetes Mellitus Tipo 2/genética , Hiperglucemia/genética , Hiperinsulinismo/genética , Obesidad/genética , Animales , Arginina/farmacología , Metabolismo Basal , Glucemia/metabolismo , Presión Sanguínea , Peso Corporal , Mapeo Cromosómico , Modelos Animales de Enfermedad , Glucosa/farmacología , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Lípidos/sangre , Sitios de Carácter Cuantitativo , Ratas , Ratas Endogámicas/genéticaRESUMEN
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.
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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
OBJECTIVE: The biobreeding diabetes-prone (BBDP) rat spontaneously develops type 1 diabetes. Two of the genetic factors contributing to this syndrome are the major histocompatibility complex (Iddm1) and a Gimap5 mutation (Iddm2) responsible for a T-lymphopenia. Susceptibility to experimentally induced type 1 diabetes is widespread among nonlymphopenic (wild-type Iddm2) rat strains provided they share the BBDP Iddm1 allele. The question follows as to whether spontaneous and experimentally induced type 1 diabetes share susceptibility loci besides Iddm1. Our objectives were to map a novel, serendipitously discovered Iddm locus, confirm its effects by developing congenic sublines, and assess its differential contribution to spontaneous and experimentally induced type 1 diabetes. RESEARCH DESIGN AND METHODS: An unexpected reduction in spontaneous type 1 diabetes incidence (86 to 31%, P < 0.0001) was observed in a BBDP line congenic for a Wistar Furth-derived allotypic marker, RT7 (chromosome 13). Genome-wide analysis revealed that, besides the RT7 locus, a Wistar Furth chromosome 8 fragment had also been introduced. The contribution of these intervals to diabetes resistance was assessed through linkage analysis using 134 F2 (BBDP x double congenic line) animals and a panel of congenic sublines. One of these sublines, resistant to spontaneous type 1 diabetes, was tested for susceptibility to experimentally induced type 1 diabetes. RESULTS: Both linkage analysis and congenic sublines mapped a novel locus (Iddm24) to the telomeric 10.34 Mb of chromosome 8, influencing cumulative incidence and age of onset of spontaneous type 1 diabetes but not insulitis nor experimentally induced type 1 diabetes. CONCLUSIONS: This study has identified a type 1 diabetes susceptibility locus that appears to act after the development of insulitis and that regulates spontaneous type 1 diabetes exclusively.
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Mapeo Cromosómico , Diabetes Mellitus Experimental/epidemiología , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Animales , Diabetes Mellitus Experimental/genética , Modelos Animales de Enfermedad , Ratas , Ratas Endogámicas BB , Telómero/genéticaRESUMEN
Genetic studies in experimental crosses derived from the inbred Goto-Kakizaki (GK) rat model of spontaneous diabetes mellitus have identified quantitative trait loci (QTL) for diabetes phenotypes in a large region of rat Chromosome (RNO) 1. To test the impact of GK variants on QTL statistical and biological features, we combined genetic and physiologic studies in a cohort of F(2) hybrids derived from a QTL substitution congenic strain (QTLSCS) carrying a 110-cM GK haplotype of RNO1 introgressed onto the genetic background of the Brown Norway (BN) strain. Glucose intolerance and altered insulin secretion in QTLSCS rats when compared with BN controls were consistent with original QTL features in a GK x BN F(2) cross. Segregating GK alleles in the QTLSCS F(2) cross account for most of these phenotypic differences between QTLSCS and BN rats. However, significant QTL for diabetes traits in both the QTLSCS and GK x BN F(2) cohorts account for a similar small proportion of their variance. Comparing results from these experimental systems provides indirect estimates of the contribution of genetic interactions and environmental factors to QTL architecture as well as locus and biological targets for future post-QTL mapping studies in congenic substrains.
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Animales Congénicos , Mapeo Cromosómico , Cruzamientos Genéticos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Sitios de Carácter Cuantitativo , Animales , Femenino , Masculino , Ratas , Ratas Endogámicas BNRESUMEN
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.
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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
Over the past decades, genetic studies in rodent models of human multifactorial disorders have led to the detection of numerous chromosomal regions associated with disease phenotypes. Owing to the complex control of these phenotypes and the size of the disease loci, identifying the underlying genes requires further analyses in new original models, including chromosome substitution (consomic) and congenic lines, derived to evaluate the phenotypic effects of disease susceptibility loci and fine-map the disease genes. We have developed a relational database (MACS) specifically designed for the genetic marker-assisted production of large series of rodent consomic and congenic lines ("speed congenics"), the organization of their genetic and phenotypic characterizations, and the acquisition and archiving of both genetic and phenotypic data. This database, originally optimized for the production of rat congenics, can also be applied to mouse mapping projects. MACS represents an essential system for significantly improving efficiency and accuracy in investigations of multiple consomic and congenic lines simultaneously derived for different disease loci, and ultimately cloning genes underlying complex phenotypes.