Identifying Genes

1. Identifying Genes

2. Genes for Complex Traits • Polygenic • Quantitative Trait Locus (QTL) • Need molecular genetics to identify • Multiple genes involved for trait • Additive contribution from individual genes • E.g., height, skin colour, autism, cancer • QTLs probabilistic risk factor for disorder

3. QTLs • Not necessarily genes themselves • Stretches of DNA closely linked to genes underlying the trait in question • QTLs often on different chromosomes • Could be: • many genes, each of small effect • a few genes of large effect

4. Inheritance • Mendelian inheritance at many loci • Normally-distributed trait • If n (loci involved) sufficiently high with binomial expansion (a+b)2n • Multifactorial inheritance • When 2+ genes involved in trait, with or without environmental activators

5. Genetic Mapping • Based on principle of genetic linkage • Particular loci or alleles are jointly inherited • Loci on same chromosome • Physically connected • Tend to segregate together in meiosis, so “linked”

6. Crossing Over • Where parts of chromosomes “swap” (recombination) • Separates genes on same chromosome • The further apart the gene loci, the greater the chance of separation through crossover • Relative distance between two genes used to calculate degree of linkage

7. Linkage Mapping • Alfred Sturtevant • By calculating number of recombinants get measure of distance between genes • Genetic map unit (m.u.) or “centrimorgan” (cM) • Distance between genes for which there is a 1% chance of recombination due to crossover in a generation • 1 cM is about 1 million base pairs in humans

8. Linkage Map • Shows positions of known genes and/or markers relative to each other in terms of recombination frequency • RF of 1% is equal to 1 m.u. • Greater the frequency of recombination, the further apart the markers are

9. Single-Gene Linkage • Use family pedigrees • Cotransmission of marker allele and trait (e.g., disorder) can be traced

10. Hypothetical Linkage Study

11. Actual Pedigree Data • Portion of Venezuelan HD pedigree • Illustrates inheritance of HD gene on a chromosome bearing the C haplotype at the G8 locus • A single individual (arrow) representing a recombination event was found in this portion of the pedigree

12. Affected Sib-Pair Linkage Design • Use families with two affected siblings • Share 0, 1, or 2 alleles for DNA marker • If marker not linked to gene, probabilities are 25%, 50%, and 25% for sharing 0, 1, or 2 alleles, respectively • If marker is linked, more than 25% of affected sib pairs will share two alleles for marker

13. Candidate Genes • Allelic association studies • Gene that may influence the development of a disease or trait of interest • Positional candidate genes selected for association studies based on their location in a genomic region linked to the trait of interest

14. Whole Genome Scan Genetic Map Reference Markers Physical Map Distribution of SNPs Chromosome 1 • Start with genome scan of 400 to 1000 markers spread across all chromosomes • Markers are a “dragnet” • Pinpoint location of disease genes • Human chr. 1 with different types of genetic markers overlaid on the map

15. Dense Mapping • Initial genomic scan carried out at low marker density • Next, identify chromosome regions which may be linked to disease of interest • Investigate by typing more markers (dense mapping) • This is where SNPs are becoming popular and useful • Etc.

16. Limitations • May not be possible to select potential candidate genes for some disorders • Functional candidate genes selected for association studies if it seems plausible that they influence the disorder • But: • Large number of possible candidate genes • Functional candidates might not really be involved • Expensive to carry out dense mapping

17. QTL Mapping • Determine degree of association of specific region on genome to inheritance of trait of interest • If high, region probably has something to do with trait expression • Remember, QTLs for trait can be on multiple chromosomes

18. LOD Score • Logarithm (base 10) of Odds • Statistical test for linkage analysis • Steps • Establish pedigree • Estimate recombination frequency • Calculate LOD for each estimate • Estimate with highest LOD considered best estimate • LOD score greater than 3.0 considered evidence for linkage • 3.0 means likelihood of observing pedigrees if two loci not linked is less than 1 in 1000

19. QTL-Scan for Genome 1,100 microsatellite markers for patients with osteoporosis used for genome-wide scan. <biology.plosjournals.org/perlserv/?request=slideshow&type=figure&doi=10.1371/journal.pbio.0000069&id=3630>

20. QTL-Scan on Single Chromosome A. chromosome 20 scan. B. Detail of linkage peak showing microsatellite markers (STRs), SNP, and gene locations. <biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0000069>

21. Markers • Originally, detectable phenotypes from coding DNA sequences used eye colour, protein production, etc. • More recently, noncoding DNA sequences, microsatellites, fragment length polymorphisms (e.g., AFLPs, RFLPs), etc. used

22. Genetic Marker • Really, any known DNA sequence • An observable variation in genomic loci • Mutation, alteration • Short sequence • E.g., single nucleotide polymorphism • Long sequence • E.g., microsatellites

23. Requirements • Easily identifiable • Associated with specific locus • Highly polymorphic

24. Detection • Co-dominant markers • Analyze one locus at a time • Dominant markers • Can analyze many loci at once • Direct detection by RNA sequencing • Indirect detection using allozymes • Variant forms of an enzyme coded by different alleles at the same locus

25. Amplified Fragment Length Polymorphisms (AFLPs) • Restriction enzymes cut genomic DNA • Subset of fragments then amplified • Highly sensitive method for detecting DNA polymorphisms • High resolution and sensitivity at whole genome level • Can amplify 50-100 fragments at once • Don’t need to know prior sequencing information Electropherogram trace from AFLP analysis <en.wikipedia.org/wiki/Image:Electropherogram_trace.jpg>

26. Microsatellites • Also called simple sequence repeats (SSRs) • Polymorphic loci consisting of 1-4 base pairs repeating 10-100 times • Serve as excellent gene markers • Common throughout genome and specific sequences closely linked to particular loci • Can be amplified through PCR

27. Polymerase Chain Reaction (PCR) • DNA polymerase used to amplify piece of DNA by in vitro enzymatic replication • DNA polymerase, an enzyme, adds free nucleotides to 3’ end of newly-forming strand • Get millions of copies of DNA piece • Gene, part of a gene, non-coding sequence • PCR can be modified to perform specific genetic manipulations

28. Transgenic Organism • Genetically modified organism • DNA from different sources is combined in vitro, then transferred to live organisms • Causes expression of modified or novel trait • Can study effects of genes with unknown function on phenotypic traits • Also used to make money… GloFish: first GMO sold as pets <www.glofish.com/images/glofish_005.jpg>

29. Terms • Chimera • Organism having two or more different populations of genotypes in cells originating from different zygotes • Genetic mosaic • Two populations of cells with different genotypes in single organism developed from single fertilized egg • Usually mutation during development; only affects subset of adult cells • Intersex condition: some cells XX, others XY • Trisomies from nondisjunction in early mitosis Calico cats: gene for fur colour on X chromosome; express both dominant (orange) and recessive (black) alleles <en.wikipedia.org/wiki/Image:Curlycat02.jpg>

30. Knockout • Useful for learning effects of genes that have been sequenced but have unknown or incompletely understood function • Gene is made inoperative • Compare knockout organism with normal

31. Technique • Isolate/identify gene • New DNA sequence of gene is engineered (it will be inoperable) • Typically also include marker gene (e.g., flurorescence) • Isolate stem cells from blastocyst and introduce new DNA sequence • Insert modified stem cells back into a blastocyst and allow embryo to develop • Organisms will be a chimera; parts of their bodies will be from their original stem cells, other parts from engineered stem cells

32. Effects • The knockout gene is permanently inoperable • Can examine for phenotypic changes • Assess functionality of gene

33. Limitations • Certain percentage of gene knockouts are developmentally lethal • Embryo can’t develop to adulthood • Some genes have different effects at different developmental points • May not produce observable change

34. Your Friend, the Transgenic Mouse • Science of Transgenics, Part 1 • Science of Transgenics, Part 2

35. Knockdown • GMO with reduced expression of one or more genes • Typically refers to temporary effect from using siRNA or RNAi: “gene silencing” • Degrades mRNA transcript or blocks mRNA translation • RNA Interference video

36. Knock-In • Specific allele, locus, or base sequence is manipulated to observe specific functions within the gene • Typically leads to over expression of some trait • Allows study of effect upon morphology and physiology of organism

37. Non-human Animal Models • Have the advantage of being able to induce polymorphisms • Many non-human animal genes are homologous to those of humans • Can serve as effective controlled models for human comparison at a variety of levels • Cellular to organ to organisms

38. Dictyostelium discoideum • Social amoebae • Grow as separate independent cells • Interact to form multicellular structures under adverse environmental conditions • Useful for studying genetic regulation of fundamental cellular processes • Cytokinesis, motility, phagocytosis, chemotaxis, cell sorting, pattern formation, cell-type determination <dictybase.org/Multimedia/chemotaxis/ColorcAR.avi> <dictybase.org/Multimedia/LarryBlanton/dev.html> <dictybase.org/Multimedia/LarryBlanton/culmBW.html>

39. Applications • Many issues of human health and disease depend upon individual cell behaviour • E.g., cytokinesis is critical in cell proliferation; integral part of immune response, tissue maintenance, and cancer • E.g., chemotaxis important in inflammation, arthritis, asthma

40. Molecular Genetics • Genomic sequencing underway • 6 chromosomes, 10,000+ genes • Several thousand genes have homologues to higher eukaryotes • Over 400 genes identified so far for cell motility, signal transduction, cell differentiation D. discoideum genomic information from chromosome 1

41. Caenorhabditis elegans • Nematode (round) worm • 1 mm long • 959 cells (302 nerve cells) • About 19,000 genes, half of which have been identified by purpose <www.nih.gov/science/models/c_elegans/worm.jpg>

42. C. elegans Neurons • Cells visible during development • Neuronal pathways of many behaviours known • Genetic regulation determinable through knockout, etc.

43. Observation in the Living System • Can visualize gene expression in living worms • Links gene expression to identified neurons • Expression of fusion between mec-4 and GFP (highlights touch cells) <From Rankin (2002)>

44. Mice • Main mammalian species for mutational screening • Hundreds of mutant mice strains • NIH Knockout Mouse Project (KOMP) • Mouse embryonic stem cells containing a null mutation in every gene in mouse genome Homozygous for piebald spotting allele of endothelin receptor type B.<www.informatics.jax.org/>

45. Flint et al. (1995) • Utilized allelic associations to study QTLs in mice • Open field activity (OFA)levels • F2 mice derived from cross between high and low lines selected for OFA; subsequently bred for 30+ generations • Each F2 mouse has unique allele set from original parental strain • One recombination, on average, per chromosome inherited from F1 strain

46. Shuffling Markers F2 intercross and recombinant inbred strains. Two inbred strains (shown as two chromosome pairs, black and white) are crossed to form an F1, and then intercrossed to form an F2 to one of the parental strains. Because the parental strains are inbred there will be just two alleles segregating at each locus in the F2, one from each parental strain, and consequently the genotype at any locus in the F2 must be one of AA, BB or AB. Recombinant inbred strains are generated by brother-sister mating a pair of F2 animals and inbreeding for at least 20 generations until the animals are homozygous at all autosomal loci.

47. QTL Mapping

48. Other QTL Depictions