1. Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

2. Alcaptonuria Rare disease Blackening of urine on exposure of air Alcapton or homogentisic acid (2, 5-dihydrophenylacetic acid) Alcapton is black (normally metabolized to colorless) But…only shows up in families with consanguinity “Inborn Errors of Metabolism” Recessive Mendelian

3. So how are we going to use genetics to study humans? Phenotypes are too complicated (and ambiguous) The breakthrough came with the realization that DNA markers would be useful

5. Two common types of DNA variants

6. There are no dominance and recessiveness issues All of the markers are co-dominant They are always present in every generation PCR is relatively cheap The expense is in finding the polymorphic markers

7. Offspring get EITHER A1 or A2 from Dad MENDEL’S FIRST LAW Offspring getting A1 from Dad can get EITHER B1 or B2 from Dad MENDEL’S SECOND LAW Dad heterozygous at genes A, B and C Mom heterozygous at genes A, B and C (and different from Dad) Offspring get only B1 and C1 Or B2 and C2 from Dad LINKAGE

8. Recall: Mapping in Drosophila Ly Sb br + + + + + br

9. Mapping in humans just like any other organism Distance in cM = # recombinant gametes/Total Two Problems…. The Major Problem: Low Numbers The not quite as major problem: The “Phase”

10. Sir Ronald Fisher, 1890 - 1962 Dealing with low numbers…..

11. The solution comes from the “Likelihood Ratio” Consider two genes, and at least 1 heterozygous parent Analyze the genotypes of the family members Consider some recombination frequency  L  = Likelihood that the genotypes arose from linked genes given  Likelihood that the genotypes arose due to segregation of unlinked genes

12. Dd 12 dd 22 dd 12Dd 22 dd 12 If linkage, was Mom: D 2 d 1 D 1 d 2 Dd 12 4 Parental 1 Recombinant 1 parental 4 Recombinant OR Mom is “Informative” Dad is “Non-informative”

13. L  = [(0.9) 4 (0.1) + (0.1) 4 (0.9)]/ 2 D 2 d 1 D 1 d 2 4 Parental 1 Recombinant 1 parental 4 Recombinant OR (0.5) 5 Calculate the likelihood given both phases and then average Z  (the lod score..log of odds) = log 10 L  = 0.021

14. L  = [(0.9) 5 + (0.1) 5 ]/ 2 (0.5) 5 Z  = log 10 L  = 0.97 A lod score of 0.97 means the genotypes are 10 times more likely to arise by segregation of linked genes (  =0.1) than by segregation of unlinked genes So crossover has a penalty and lowers the lod score (0.021<0.97) What if there were no crossovers?

15. L  = [(0.8) 4 (0.2) + (0.2) 4 (0.8)]/ 2 D 2 d 1 D 1 d 2 4 Parental 1 Recombinant 1 parental 4 Recombinant OR (0.5) 5 Z  = log 10 L  = -0.549 (note lod scores can be negative) What if we calculate for  = 0.2 For no crossovers at  of 0.2 Z  = log 10 L  = 0.72

16. Therefore….. The lod score varies with  The lod score varies with data (no crossovers vs 1 crossover) What should we expect? What should be our confidence? Z  = 3 This is when it is 1000 times more likely that genotypes arose from linked genes than from unlinked genes

17. Calculate the lod score for any two markers at every  and plot the results

18. Mapping DNA markers The principle is the same except that the markers are co-dominant We saw before that markers are “informative” in meiosis when doubly heterozygous The utility of any marker will depend on the degree of heterozygosity in the population For best microsatellites the heterozygosity ranges from 10-30% Need sufficient number to cover the human genome The human genome is ~3000 cM in genetic distance For = 0.01 (1 cM resolution) you need 3000 evenly spaced informative markers First step is to identify as many microsattelite markers as possible You have no idea, a priori, where thy map relative to each other. So Map them! Where do we get the DNA for doing the mapping?

19. CEPH Centre d'Etude du Polymorphisme Humain Maintain the DNA database of ~10,000 informative microsattelite markers DNA from each individual of 50 large 3-generation families Generated immortalized lymphocytes from each individual (cell lines) Represents a renewable resource of DNA Computed 25,924,717 lod scores between all pairs of marker loci on each pair of segregating chromosomes From these data, and the principles of lod score mapping we generate the human genetic map

20. Note that the genetic map is different for men and women Recombination frequency is higher in meiosis in women