sir archibald e garrod 1857-1936 1902 – alcaptonuria – black urine - (madness of king george)

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

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

Sir Archibald E Garrod 1857-1936

1902 – alcaptonuria – black urine - (Madness of King George)

Page 2: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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

Page 3: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

So how are we going to use genetics to study humans?

Phenotypes are too complicated (and ambiguous)

The breakthrough came with the realizationthat DNA markers would be useful

Page 4: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)
Page 5: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

Two common types of DNA variants

Page 6: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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

Page 7: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

Offspring get EITHERA1 or A2 from Dad

MENDEL’S FIRST LAW

Offspring getting A1 fromDad can get EITHER B1 orB2 from Dad

MENDEL’S SECOND LAW

Dad heterozygousat genes A, B and C

Mom heterozygousat genes A, B and C(and different from Dad)

Offspring get only B1 and C1Or B2 and C2 from Dad

LINKAGE

Page 8: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

Recall: Mapping in Drosophila

Ly Sb br

+ + +

+ + br

+ + br

Page 9: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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”

Page 10: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

Sir Ronald Fisher, 1890 - 1962

Dealing with low numbers…..

Page 11: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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

Page 12: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

Dd 12 dd 22

dd 12 Dd 22 Dd 22 dd 12

If linkage, was Mom:D

2

d

1

D

1

d

2

Dd 12

4 Parental1 Recombinant

1 parental4 Recombinant

OR

Mom is “Informative” Dad is “Non-informative”

Page 13: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

L = [(0.9)4 (0.1) + (0.1)4 (0.9)]/ 2

D

2

d

1

D

1

d

2

4 Parental1 Recombinant

1 parental4 Recombinant

OR

(0.5)5

Calculate the likelihood given both phases and then average

Z (the lod score..log of odds) = log10 L = 0.021

Page 14: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

L = [(0.9)5 + (0.1)5 ]/ 2

(0.5)5

Z = log10 L = 0.97

A lod score of 0.97 means the genotypes are 10 times more likely to ariseby 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?

Page 15: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

L = [(0.8)4 (0.2) + (0.2)4 (0.8)]/ 2

D

2

d

1

D

1

d

2

4 Parental1 Recombinant

1 parental4 Recombinant

OR

(0.5)5

Z = log10 L = -0.549 (note lod scores can be negative)

What if we calculate for = 0.2

For no crossovers at of 0.2 Z = log10 L = 0.72

Page 16: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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 fromlinked genes than from unlinked genes

Page 17: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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

Page 18: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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?

Page 19: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

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

Page 20: Sir Archibald E Garrod 1857-1936 1902 – alcaptonuria – black urine - (Madness of King George)

Note that the genetic mapis different for men and women

Recombination frequency is higher in meiosisin women