Understanding quantitative genetic variation.

1. Understanding quantitative genetic variation. (1) QTL analysis of model organisms and farm animals. (2)Twin studies.

2. Francis Galton, Karl Pearson, WFR Weldon, representatives of ‘biometric school: - established at 19th century the bases of quantitative genetics and statistical tools of correlation and regression ‘biometric view’ placed emphasis on natural selection acting on minor variants as opposite to Mendelian genetics At the beginning of 20th century it was recognized that minor Mendelian variants could account for inherited variation of continuous traits Quantitative traits: (1) morphological traits (height, body mass, skeletal shape, (2) metabolic traits (hypertension, drug response), (3) survival traits (longevity, immunity) etc.

3. Quantitative traits: (1) a few loci with large effects or (2) Many loci with small effects? Infinitesimal model: Quantitative trait variation is based on very many genes of very small effect Thus, selection does not cause appreciable changes in frequency of any particular allele and doe not erode variation, which allows the response to selection to continue

4. 400 unlinked loci with minor effects, incl 2 major loci 100 unlinked loci, allele frequencies as in (a) 10 unlinked loci with major alleles Two simulation replicates 2 major loci fixed Time in generations Alternative genetic models for long-term selection response. Simulations of artificial selection on an addictive trait with environmental variance Ve=1. 50 highest scoring individuals were chosen from 250 in each generation. Initial h2=30%

5. QTL analysis: *Localization of regions where loci responsible for quantitative trait variation lie by linkage analysis *this analysis is done in experimental organisms by crossing two lines that differ markedly in the quantitative trait and also differ in alleles at marker genes or loci *F1 between the two lines is then crossed with itsself to make a segragating F2, or it may be back-crossed to one of parental lines. Receiver’s chromosomes Donors’s chromosomes X F1 Repeated backcrossing of F1 Animals to receiver strain Intercrosses of F1 animals Brother-sister inbreeding Recombinant inbred strain Consomic Congenic

7. Advantages of rodent models: *numerous genetically well-defined lines that differ from each other in phenotypic characteristics *modest cost of mainenance *short generation times *can be kept under well defined environmental conditions *can be manipulated experimentally (transgenic, knockout) *interraction of genes and environment is expressed in measurable phenotypes *several naturally occurring models and genetic toolsline congenic etc. strains *rodent-human homology well understood Other animal models: Dogs (behaviour), Pigs (atherosclerosis), baboons (Parkinson disease), zebrafish and Ciona intestinalis (developmental biology)

8. Cox and Brown, 2003

9. History of rat research: *The first mammalian species to be domesticated for scientific work (19850ies) *the first genetic studies were carried out 1877-1885 by Crampe and were focused on the inheritance of coat colour *in 1903 William Bateson used the consepts of Mendel’s laws to show that a variant of a rat coat-colour gene was inherited in a Mendelian fashion. *the first inbred strain was established by King 1909, the same year that inbreeding began for the first inbred strain mouse DBA. *Mouse soon became the model of choise for mammalian geneticists, while rat became a model for physiologists, nutritionists, and other biomedical researchers *234 inbred rat strains have been developed primarily by physiologists by selective breeding for disorders ranging from hypertension to urological defects *1sr trangenic rat 1990

10. Integrating data from the rat and mouse for studies of human disease Transgenic mouse: harbour large pieces of human genome Knockout mouse: deletion of a syntetic locus to human disease in mouse genome using homologous recombination

11. NB! Rodent models do not always recapitulate the outcome of human disese, owing species specific differences Nethertheless, the animal models can provide the entry points into clinically relevant pathways e.g. Both, in mice and rats involvement of LEPTIN in obesity has been shown. There are conflicting result in involvement of leptin in human obesity BUT elucidation of the leptin pathway has provided new insights into food intake and metabolism that before these studies were unknown both in human and rodents

12. QTL mapping in inbred rodent strains versus single gene knock-out or transgenic animal • + can identify regions of the genomic that carry allelic variants that collectively contribute to certain phenotype • + quicker results • + closer to the human phenotype of a complex disease • a QTL is genetically complex and large region • combinatory effect of several genes, difficult to decide what is primary and what is secondary effect • hitchhiking variation of linked genes, difficult to decide the causative variant • Application of congenic, recombinant congenic and consomic strais

13. Models for the interraction of loci in complex diseases (by Gabriel et al., 2002) GENOTYPE PHENOTYPE GENOTYPE PHENOTYPE EFFECT 1. Additive 3. Epistatic EFFECT + + individual no individual effect 4. Mixed Multipli- cative 2. Multipli- cative x x OR x x In combination only either combination only

14. Recombinant congenic strain generation: Two parental strains are crossed An offspring F1 is back-crossed to one of the parents Repeated back-crossing to the blue strain accumulates recombination events, reducing the amount and length of green inherited DNA (results in CONGENIC strains) Finally the strains are inter-crossed several times, brother-sister mated, and the green DNA fragments fixed to homozygosity on a blue DNAa background (results in RECOMBINANT CONGENIC STRAIN)

15. QTL study from New Zealand Obese (NZO) and Nonobese Nondiabetic (NON) mouse strain (Reifsnyder P et al, 2002): Used Recombinant Congenic approach developing 10 interval directed strains that carry QTL interval DNA from NZO on a relatively lean NON background. Important observations: Computationally predicted epistatic interractions between QTLs from studies of F2-intercrosses and backcrosses were confirmed Obesity could be uncoupled from diabetes (but not vice verse) Stepwise diabetes development with step-wise increase in diabetogenic QTL was shown Toxisity and vialibity effects were separated and investigated The map position of QTL-containing loci were refined

16. Functional complementation by transgenesis:The role of a candidate gene in a quantitative trait locus (QTL) that is carried by a congenic rat strains on a reciprocal genetic background can be tested by transgenesis. The copy number control is required to rule out the likelihood that the phenotypic effects of the transgene have been brought about by introduction of an abnormal number of gene copies Jacob and Kwitek, 2001

17. Mutagenesis screens in rodents *New mutations can be generated in mice at relatively high frequencies using radiation, chemical mutagens and transgene insertion. *powerful germline mutagen ENU (N-ethyl-N-nitrosourea) *ENU is a point mutagen that is capable of inducing many different alleles *ENU can induce mutations in mouse spermatogonia at rate equivalent to isolating a mutation in a single gene of choise in 1 every 700 gametes screened *typical protocol includes the treatment of male mice with ENU, followed by mating with wild-type mice.

18. Comparative genomics Questions: Broad insite about types of genes can be gleaned by genomic comparisons at very long phylogenetic distances, e.g. > 1 billion years since their separation e.g. by comparing genomes of yeast, worms and flies reveals that these eukaryotes encode many of the same proteins At moderate phylogenetic distances (70-100 million years) both functional and nonfunctional DNA is found within the conserved DNA e.g. comparison between mouse and human, among several species of yeast or among enteric bacteria Here functional sequences show signal of puryfing (negative) selection. These comparisons assist to identify coding exons, noncoding RNAs, concerved regulatory regios Comparison between close species as human and chimpanzee (separated about 5 million years), are particularly apt for finding the key sequence differtences in the organisms. Here the sequences have changed under positive selection.

19. Conserved syntety between human and mouse Human and mouse comparison: *Separated 75-80 million ya *the large-scale gene organization and gene order have been preserved *about 90% of human genome is in large blocks of homology with mouse

20. QTL associated with complex disease phenotypes that map to regions of conserved syntety in the human, rat and mouse genomes. Blue -obesity, red - hypertension, autoimmune/inflammatory phenotype

21. QTL associated with complex disease phenotypes that map to regions of conserved syntety in the human, rat and mouse genomes. Blue -obesity, red - hypertension, autoimmune/inflammatory phenotype

22. Farm animals as models of quantitative genetics: *large family sizes *Monitored long for thousands of years by human *large variety of different breeds, which have been adapted for different needs and climate conditions *rich collection of mutation that affects phenotypic traits *possible to dissect monogenetic traits as coat colour, but also qualtitative traits as fertility, growth, behaviour

24. An intercross between the european wild board (recedssive allele of KIT locus) and Large white Domestic pigs (dominant allele of the KIT locus. The segregation of white colour in F2. The three KIT alleles in the pig. The tandem duplication present in Patch and Dominant White alleles is larger than 400 kb and it includes the entire KIT sequence KIT - mast/stem-cell growth factor receptor; crucial for the survival of migrating melanocytes during embryogenesis and for hematopoiesis and germ-cell development Splice mutation GtoA: Skipping of exon 17 Johansson Moller et al, 1996; Marklund et al, 1998

25. Farm animal breeds are expected to have large homozygous regions due to selective sweeps: Advantage in mapping genes responsible for the trait Andersson and Georges, 2004

26. *Q1 to Q2 mutation at certain at generation 0: complete LD between the Q2 and alleles of other loci at mutated gamete LD is decayed through generations by recombination events Haplotype of closely linked loci has preserved around Q2 Identity by decent mapping (IBD) - determining the minimum shared haplotype among the animals carrying Q2 allele. Andersson and Georges, 2004

27. Approaches to mapping and positional cloning of QTL in domestic animals (Andersson and Georges, 2004)

28. Andersson and Georges, 2004

29. Example: IGF2 quantitative trait locus in pigs *Idenification of IGF2 by intercrosses between European wild board and Large White domestic pigs *QTL from domestic pig was associated with high muscularity, less backfat and a larger heart *Swedish Large White and Pietrain pigs shared haplotype IDB: high muscle growth (IGF*Q) *wild board and Belgian Large White: alternative haplotype with low muscle growth (IGF*q) *Belgian Large White actually as a hybrid between European and Asian breeds, thus possessing IGF*q *the causative variant of IGF*Q is G-to-A substitution at position 3072 in intron 3

30. silencer mutated silencer 3 -fold upregulation of IGF2 expression in postnatal, but not prenatal scelectal and cardiac muscle Van Laere et al., 2003

31. Revival of twin studies Galton F (1875) “The history of twins as a criterion of the relative powers of nature and nurture” JR Anthropol Inst Gt Br Ireland 5, 391-406. *the first classical twin paper Siemens HW (1924) Die Zwillingpathologie:Ihre Bedeutung, ihre Methodik , ihre bisherigen Ergebnisse. Springer, Berlin *the twin rule of pathology: any heritable disease will be more CONCORDANT in identical twins than in non-identical twins, and concordance will be even lower in non-siblings.

32. Classical twin studies: *Compares phenotypic resemblance of monozygotic (MZ) and dizygotic (DZ) twins *MZ twins derive from single fertilized egg and therefore inherit identical genetic material *Hertitability of the trait h2 =2x difference in MZ and DZ correlations e.g. for depression rMZ=0.4 and rDZ=0.2, h2 =40% *Contribution due to shared environment is calculated rMZ- h2 and rDZ- h2/2 e.g. for taking up smoking rMZ=0.9 and rDZ=0.7 Environmental role= 0.9-0.4 =0.7-0.2+= 50%

33. X X X X age Percentages of variances explained by: Genetic factors Environmental factors Differences in age Unique environmental influence X Differences between Males and females EXAMPLES OF CLASSICAL TWIN STUDIES

34. Boomsma, 2002

35. Boomsma, 2002

36. Twin discordance: MZ twins - only one exhibits cleft lip and palate Different penetrance? * Patients in father’s family

37. Twin registers: • *originate as spin-offs from specific research projects, usually in medicine or psychology • GENOMEUTWIN - european community funded large integrated project involving 6 participating twin cohorts from Scandinavia, the Netherlands and Italy • More than 0.6 million pairs of twins • More than 30 000 DNA samples

39. Twin registers outside Europe: USA (13), Canada (2), Australia (3), Sri Lanka (1), China (1), Japan (1), S. Korea (2)