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Congenic-Based Strategies for Dissecting the Architecture of QTL and their Biological Functions

Congenic-Based Strategies for Dissecting the Architecture of QTL and their Biological Functions Dominique Gauguier, Stephan C Collins, Karin Wallace, Robert H Wallis, Steven P Wilder The Wellcome Trust Centre for Human Genetics University of Oxford

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Congenic-Based Strategies for Dissecting the Architecture of QTL and their Biological Functions

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  1. Congenic-Based Strategies for Dissecting the Architecture of QTL and their Biological Functions Dominique Gauguier, Stephan C Collins, Karin Wallace, Robert H Wallis, Steven P Wilder The Wellcome Trust Centre for Human Genetics University of Oxford http://well.ox.ac.uk/rat_mapping _resources/

  2. F0 Disease Susceptible Disease Resistant F2/BC Mapping Disease Susceptibility Genes in Rodents Genetic mapping Positional cloning Disease gene identification

  3. F0 Disease Susceptible Disease Resistant F2/BC F0 Disease Susceptible Disease Resistant F2/BC Genetic Analysis of Quantitative Traits in Inbred Rodent Models Mapping Disease Related Susceptibility and Modifier Loci

  4. QTL Mapping in Rodent Models of Human Complex Traits Inbred strains Disease models Genetic makers and dense linkage maps Gene maps Genomic sequence Comprehensive in vivo phenotyping Disease onset Disease progression Fine pathophysiological profiling

  5. Weeks Genetic Dissection of Complex Phenotypes in Rodents Models of Human Diseases Absence Epilepsy 24h-EEG Recording Hypertension Blood pressure recording WAGxACI RNO5 RNO9 Van Luijtelaar, Cox, Gauguier

  6. Cross-Talk Between Tissues in the Regulation of Glucose Metabolism Saltiel and Kahn, Nature 414, 799-806, 2001

  7. Cross-Talk Between Tissues in the Regulation of Glucose Metabolism Type 2diabetes Obesity Hypertension Dyslipidaemia

  8. Identification of Diabetes Susceptibility Genes in Rodent Models • What is the contribution of genetic factors in the control of quantitative variations of • diabetes related phenotypes in rodent models and what are their significance? • How can we efficiently refine the localisation of diabetes gene(s) at a QTL? • What is the most critical criterion for disease gene identification? • QTL architecture • Refined QTL map location • QTL pathophysiological profile • What is a diabetes candidate gene / gene pathway? • What is not a diabetes candidate gene / gene pathway? • How can we translate genetic results obtained in rodents to human diabetes?

  9. Glucose Homeostasis 6 Adiposity Index 5 4 Lod score 3 2 1 0 5cM Genetic Mapping of a QTL Linked to Glucose Tolerance and Adiposity in Rat Chromosome 1 in a GKxBN F2 Cross

  10. Fasting Post abs. Acute Glucose Glucose Insulin Insulin Acute Insulin Adiposity Glycaemia Glycaemia Response to Glucose Tolerance Sensitivity Secretion Secretion Linkage Mapping of Diabetes Related Phenotypes to Rat Chromosome 1 in a GKxBN F2 Cross Locus Nidd/gk1 LOD score

  11. Role of Statistical and Functional Investigations in the Resolution of QTL Complexity Fine QTL mapping in experimental crosses and hybrid populations (RCS, RIS) Increase genome coverage Increase population size Breed for several generations Fine QTL functional characterisation in congenics Validation of original QTL phenotypic effect(s) Test for QTL interactions Test for X-independent gender differences in diabetes phenotypes Phenotype profiling Assess pleiotropy or multiple gene effects? Provide key tissue targets for gene expression studies

  12. X X GK BN 5 5 - - 6 backcross using 6 backcross using marker assisted marker assisted protocol protocol X X X Congenic sublines QTL targeted F2 cross (n=245) QTL substitution line Fine QTL mapping Phenotype profiling (100) Gene expression profiling Sequence variants analysis Functional validation of sequence variants Comparative genome analysis Human genetics QTL validation Phenotype profiling (>150) QTL validation (92 phenotypes) QTL dissection QTL robustness & significance QTL architecture Epistasis X-independent gender differences From Statistical Estimates of QTL Maps to QTL Architecture and Functional Characterisations

  13. Morphological Alterations Kidney Pancreatic islets Insulin Secretion Glucose Tolerance BN BN 300 100 280 260 80 240 220 QTL 60 QTL 200 180 40 160 140 20 120 0 0 5 10 15 20 5 10 15 20 30 30 Time (min) Time (min) Weeks Metabolic and Hormonal Profiling of QTLs in a QTL Substitution Strain Blood Pressure Phenotypes Plasma Lipids

  14. Genetic Mapping of Diabetes Related Phenotypes in 245 “F2” Hybrids Derived from a QTL Specific Congenic Strain The linkage map was constructed with 85 markers using JoinMap. ANOVA was used to test for linkage between marker loci and phenotypes adjusted for sex effects. Glucose Tolerance Glucose-Induced Body Weight Lipids (Chol, FG Arginine-Induced Insulin Resistance Insulin Secretion Organ Weight TG, LDL, VLDL, FI Insulin Secretion 3 months 6 months 3 months 6 months Phos., NEFA 3 months 6 months

  15. Genetic Mapping of Diabetes Related Phenotypes in 245 “F2” Hybrids Derived from a QTL Specific Congenic Strain The linkage map was constructed with 85 markers using JoinMap. ANOVA was used to test for linkage between marker loci and phenotypes quantified in males and females Males Females Glucose Tolerance Glucose-Induced Body Weight Lipids FG Arginine-Induced Insulin Resistance Insulin Secretion Organ Weight FI Insulin Secretion 3 months 6 months 3 months 6 months 3 months 6 months Glucose Tolerance Glucose-Induced Body Weight Lipids FG Arginine-Induced Insulin Resistance Insulin Secretion Organ Weight FI Insulin Secretion 3 months 6 months 3 months 6 months 3 months 6 months

  16. LOD score LOD score 1 2 3 4 5 6 1 2 3 4 Glucose Tolerance Glucose-Induced Body Weight Lipids FG Arginine-Induced Insulin Resistance Insulin Secretion Organ Weight FI Insulin Secretion 3 months 6 months 3 months 6 months 3 months 6 months Glucose Tolerance / Insulin Resistance Insulin Secretion Chromosomal Mapping of Diabetes Traits in a Classical F2 Cross and in a Two Generation Cross Derived from a QTL Specific Congenic Strain Classical F2 Cross QTL Specific F2 Cross Congenic Lines

  17. LOD score LOD score 1 2 3 4 5 6 1 2 3 4 Glucose Tolerance Glucose-Induced Body Weight Lipids FG Arginine-Induced Insulin Resistance Insulin Secretion Organ Weight FI Insulin Secretion 3 months 6 months 3 months 6 months 3 months 6 months Glucose Tolerance / Insulin Resistance Insulin Secretion Validation of QTL Effects in Congenic Sublines Designed for a Diabetes QTL in the Rat Classical F2 Cross QTL Specific F2 Cross Congenic Lines

  18. From Statistical Estimates of QTL Maps to QTL Architecture and Functional Characterisation • Chromosome and QTL specific congenic lines are essential new models that allow • Confirmation of the chromosomal localisation of QTL • Validation and refinement of QTL phenotypic effects • Estimations of QTL robustness and significance • Tests for interactions with known QTLs • Assessment of X-independent gender effects • Targeting specific genetic regions that can be dissected in congenic sublines • Congenic sublines subsequently designed for specific loci provide original models for • Fine QTL mapping • A Comprehensive pathophysiological profiling of gene variants • A better definition of tissue targets for expression studies • The definition of target loci for genetic studies in human • The identification of potential candidate genes and gene pathways for further investigations in rodents and human

  19. Mutation detection Enzyme activity in CHO-IR transfected cells Rat Human Proof of Concept Sequence and Functional Analysis of the Lipid Phosphatase SHIP2 in Rat and Human Marion et al., Diabetes 51:2012-2017,2002

  20. The Wellcome Trust Centre for Human Genetics University of Oxford Stephan C Collins Robert H Wallis DPhil Karin Wallace Karene Argoud Marie Therese Bihoreau PhD Pamela J Kaisaki PhD Steven Wilder Mark Lathrop, CNG, Paris http://well.ox.ac.uk/rat_mapping _resources/

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