Trinucleotide satellite lengths and AR transcriptional activity

1. Trinucleotide satellite lengths and AR transcriptional activity The androgen receptor gene contains two polymorphic trinucleotide microsatellites in exon 1. The first microsatellite (nearest the 5' end) contains 8 to 60 repetitions of the glutamine codon "CAG" and is thus known as the polyglutamine tract. The average number of repetitions varies by ethnicity, with Caucasians exhibiting an average of 21 CAG repeats, and 18 in Blacks. In men, disease states are associated with extremes in polyglutamine tract length: prostate cancer, hepatocellular carcinoma, and mental retardation are associated with too few repetitions, while spinal and bulbar muscular atrophy is associated with a CAG repetition length of 40 or more. Some studies indicate that the length of the polyglutamine tract is inversely correlated with transcriptional activity in the AR protein, and that longer polyglutamine tracts may be associated with male infertility and undermasculinized genitalia in men. A comprehensive meta-analysis of the subject published in 2007 supports the existence of the correlation.

2. How it works…

3. Population genetics, health and disease Why population genetics is important for human health and disease Basics of population genetics: the main forces, and examples How genes can contribute to disease

4. Population genetics, health and disease (1) Why population genetics is important for human health and disease (a) Human evolution has been underlain by adaptive and non-adaptive changes in allele frequencies (b) Diseases are commonly due to effects of alleles, and alleles interacting with environments - there is a spectrum from single-locus disorders to polygenic disorders

6. Some genetically-based traits that evolved in the human lineage Hair that keeps growing Blue eyes Blond hair Ability to digest milk after infancy Highly-articulate speech (6) Schizophrenia (7) Liability to Alzheimer’s (8) Menopause (9) Big brains etc etc

7. (2)Basics of population genetics: the main forces, and examples

8. Gregor Mendel (1822-84) • discovered the laws of heredity in 7 hybridization experiments on 19,959 pea plants • published his results in 1865, but they were ignored until 1900

9. Mendel’s peasMendel investigated inheritance of these characteristics: height (tall/short) pea colour (green/yellow) pea shape (round/angular) pod shape (full/pinched) pod colour (green/yellow) flower colour (purple/white) Peas, disease, its all genetically the same for single locus phenotypes

10. What’s important about Mendel • He provided evidence that inheritance is particulate, not blending • He established the distinction between inheritance and expression of genes, with regard to dominance and recessiveness (which are caused by evolved physiological-developmental effects of alleles)

11. Hardy-Weinberg Equilibrium: A Null Model to compare to your Real Data • ASSUMES: • Pretty much: No mutation, no selection, no migration (gene flow), • random mating by genotype, large population size (no random • changes due to sampling error [= genetic drift]) • PREDICTS: • If allele frequencies are p and q, then genotype frequences are p2, 2pq, q2 • alleles: A B genotypes: AA AB BB Essence of Hardy-Weinberg Equilibrium: NO POPULATION-GENETIC FORCE: NOTHING HAPPENS like black and white marbles in a jar, in pairs, then single….

12. p q p pq 2 p pq 2 q q Hardy-Weinberg equilibrium If the frequency of one allele (A) is p and that of the other allele (B) is q, random mating is random combining of gametes, which leads to (p + q)2 = p2 + 2pq + q2 Freq AA Freq AB Freq BB

13. Of what USE is the HW Equilibrium? NO POPULATION-GENETIC FORCE: NOTHING HAPPENS -can predict genotype frequencies (p2, 2pq, q2) from allele frequencies (p, q) YESIS A POPULATION GENETIC FORCE, THEN SOMETHING SPECIFIC HAPPENS AND CAN SEEK TO INFER WHAT CAUSED IT For example: Selection: changes relative frequencies of one or two of the genotypes, due to differences in relative fitness (eg survival) Inbreeding leads to more homozygotes than predicted

14. EXAMPLE Observe genotypes (number of indviduals) AA 8 AB 64 BB 128 Calculate observed genotype frequencies. NOW! I MEAN IT! Calculate observed allele frequencies. NOW! I MEAN IT! What are the genotype frequencies expected under Hardy-Weinberg Equilibrium? EASY PEASY! Compare observed with expected genotype frequencies

15. ANOTHER EXAMPLE Observe genotypes (number of indviduals) AA 20 AB 160 BB 20 Calculate observed genotype frequencies. Calculate observed allele frequencies. What are the genotype frequencies expected under Hardy-Weinberg Equilibrium? Compare observed with expected genotype frequencies Hm-m. What next?

16. HOW unlikely is it to get such results BY CHANCE? • TEST vs CHANCE using -square test • Genotype Obs Exp number of individuals AA 10 25 AB 80 50 BB 10 25 • Sum of (Obs - Exp)2 • = (10-25)2 + (80-50)2 + (10-25)2 • = 36 • Using distribution with 1 df, p < 0.001, so odds of getting this result by chance are less than 1 in 1000 Exp 25 50 25

17. What if your samples sizes were smaller? (AA: 1, AB: 8, BB: 1) Still significant? What if, in a different study, your samples sizes were very large, such as 1,000,000, compared to moderate (such as 100)? What if the same study has been conducted 19 times previously with non-significant results, but you find p = 0.0499? -false positives -false negatives -the file drawer problem -statistical compared to biological ‘significance’ Statistics and clinical trials: publish trial design, planned statistics in advance; consequences of false positives, negatives (& double-blind design, placebo effects, conflicts of interest...) STATISTICS ARE ‘TRUTH’

18. Albinism • inheritance: recessive alleles at 2 loci • incidence: 1-in-40,000 births in Europe • symptoms: no body colour, visual deficits • cause: lack of tyrosinase means that melanin can’t be synthesized

19. Doing some sums: albinism • autosomal recessive incidence of 1-in-40,000 • if dominant is p, and recessive albinism is q • q2 = 1/40,000, or 0.000025 • q = √0.000025 = 0.005 • since p+q = 1, p = 1- 0.005 = 0.995 • 2pq = 2 x 0.995 x 0.005 = 0.00995 or 0.995% • ie, ±1-in-100 is a carrier of the albinism allele • chance of carriers mating: 1-in-10,000 • chance of homozygosity: 1-in-4 = 1-in-40,000

20. The real genetic and genomic world is not A’s and peas: Human genome: about 3 billion nucleotides, with about 3 million of them variable among any two random humans (99.9% identity); most variants probably have no phenotypic effects (are ‘neutral’) Human Genome Project has provided the sequence (all online) of one human, but the most interesting and important data as regards health is the variation among humans, analyzed using the: HapMap (Haplotype Map) project has characterized genetic variation among three major populations, one African, one Asian, one Caucasian (one or more common SNP genotyped at least every 5000 base pairs); > 1 million SNPs overall 1000 Genomes project: full sequences of 1000 humans -> rare variants SNP - single nucleotide polymorphism (2 or more bases at a locus) Haplotype - linear combination of SNPs or other markers on a chromosome such as C...C....A.T (haplotype 1), C...G....A.T (haplotype 2); sets of linked bases tend to be inherited together -- form flanked ‘blocks’ Microsatellites - repetitive elements with variable numbers of short repeats such as CAGCAGCAG...or ATATAT - used as markers, and underly some diseases Copy number variation - variation in number of copies of large sections of genome, including one or more genes (large deletions, duplications)

21. Some important findings from HapMap project (and earlier studies using other genetic markers) About 10-15% of total human genetic variation is among populations; rest is within populations Africa harbours substantially higher levels of human genetic variation than other regions Patterns of natural selection ‘for’ given alleles (positive selection) vary substantially among populations -> local adaptation

24. More important facts about mutation: -Types of mutation: somatic vs germline; single base pairs, insertions/deletions repeats,rearrangements, copy number variation; in coding, non-coding, regulatory DNA -Mutations have deleterious effects in the great majority of cases, so selection should minimize the mutation rate, subject to constraints, tradeoffs (repair ability, time constraints in replication) -Many human diseases are caused by de novo mutations (eg about 10% of cases of autism may be due to de novo germ-line mutations) - these diseases can persist under a balance between mutation and selection - see OMIM for human knowledge in this area http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim -The larger the population, the greater the scope and potential for mutations to turn out to be adaptive

27. Natural Selection, for mendelian loci Only force that can cause adaptation (can also result in maladaptations) World of ‘things’, which vary in size o O o o O O o O O o o Live in an environment, have a niche U u U u u U U u Reproduce, there is heritable variation o -> o , O -> O Change in environment, U u U u u U U u selection for smaller size Evolutionary change, and o o o o o o o o o adaptation

28. Resistance to antibacterial soap Generation 1 1.00 not resistant 0.00 resistant Natural selection, a simple, general example

29. Natural selection Resistance to antibacterial soap Generation 1 1.00 not resistant 0.00 resistant

30. Natural selection Resistance to antibacterial soap Generation 1 1.00 not resistant 0.00 resistant Generation 2 0.96 not resistant 0.04 resistant mutation!

31. Natural selection Resistance to antibacterial soap Generation 1 1.00 not resistant 0.00 resistant Generation 2 0.96 not resistant 0.04 resistant Generation 3 0.76 not resistant 0.24 resistant

32. Natural selection Resistance to antibacterial soap Generation 1: 1.00 not resistant 0.00 resistant Generation 2 0.96 not resistant 0.04 resistant Generation 3 0.76 not resistant 0.24 resistant Generation 4: 0.12 not resistant 0.88 resistant Rapid evolution of adaptation by natural selection - genetic basis?

33. Natural Selection, for mendelian loci • Only force that can cause adaptation (remember the ‘things’!) • Common situation for functional sites is usually to have one allele/haplotype common (ancestral), rare mutant (derived) alleles selected against (‘purifying selection’), since mutations are usually bad - genetic situation stays same (3) Various forms of selection at one locus (AA,Aa,aa) • forrecessive mutation, fitnesses: aa > Aa, AA • fordominant mutation: fitnesses Aa, AA > aa It takes a LONG TIME for advantageous mutations to reach fixation (dozens to hundreds to thousands of generations) (all individuals, lineages with disadvantageous alleles must die) ->leads to MISMATCHES as environments, selection changes

34. More on various forms of selection at one locus (AA,Aa,aa) (c) for heterozygous genotype Aa > AA, aa -due to nature of inheritance (a constraint), maladapted homozygotes are generated every generation (d) against recessive genotype aa < AA,Aa -due to fact that vast majority of ‘a’ alleles are in heterozygotes it is exceedingly difficult for selection to remove deleterious ‘a’ allele from population For example, with p=0.95, q=0.05; q2=0.0025, 2pq=0.095 - very little variation is ‘visible’ to selection (e) against dominant genotype AA, Aa < aa -very effective at removing ‘A’ allele, UNLESS phenotypic effects manifest after age of reproduction (eg Huntington’s disease)

35. Rare Alleles and Eugenics • A popular idea early in the 20th century was “eugenics”, improving the human population through selective breeding. The idea has been widely discredited, largely due to the evils of “forced eugenics” practiced in certain countries before and during World War 2. We no longer force “genetically defective” people to be sterilized. • However, note that positive eugenics: encouraging people to breed with superior partners, is still practiced in places. • The problem with sterilizing “defectives” is that most genes that produce a notable genetic diseases are recessive: only expressed in heterozygotes. If you only sterilize the homozygotes, you are missing the vast majority of people who carry the allele. • For example, assume that the frequency of a gene for a recessive genetic disease is 0.001, a very typical figure. Thus p = 0.999 and q = 0.001. Thus p2 = 0.998, 2pq = 0.002, and q2 = 0.000001. The ratio of heterozygotes (undetected carriers) to homozygotes (people with the disease) is 2000 to 1: you are sterilizing only 1/2000 of the people who carry the defective allele. This is simply not a workable strategy for improving the gene pool. Recessive deleterious (and advantageous) alleles are present mainly in heterozygotes, ‘hidden’ from selection; most people have multiple, rare, homozygous-lethal alleles in their genome

36. Eradicating dominant disorders • Huntington’s—and any other dominant disorder—could in principle be eliminated in one generation by aborting every foetus carrying the gene • however, this would not prevent spontaneous mutations occurring (in Huntington’s, ±1 in 100,000) unless the entire population was screened for them

37. Eradicating (as best possible) recessive inherited disorders in genetic isolates Ashkenazi heritable disorders • Gaucher disease: ranges from mild to severe, sometimes treatable • Cystic Fibrosis: average life expectancy ±30 • Fanconi anemia: developmental and mental retardation, proneness to cancer • Nieman-Pick disease: fatal by age 4 • Bloom syndrome: fatal cancers by age 30 • Canavan disease: similar to Tay Sachs

38. Dor Yeshorim • Committee for the Prevention of Jewish Genetic Diseases • founded by Rabbi Josef Ekstein after losing 4 children to Tay-Sachs • community at first in denial, but later testing became widely accepted • 170,000 tested now for 9-10 diseases, ~1-in-100 couples ‘incompatible’

39. How it works • undisclosed tests carried out at school • if testees consider a relationship, they can enquire about compatibility • if only 1 is a carrier, no disclosure, but if both are, advised ‘incompatible’ and counselled • Tay-Sachs cases now almost eliminated

40. Natural selection - Lactase gene in humans Origin of animal husbandry and animal Milk as food source, less than 7000 years ago (2) Selection for lactase persistence, ability to digest milk after weaning, selects for allelic variants of LCT gene (lactase-phlorizin hydrolase)(intolerant: gassy, farty, nauseous) (3) Geographic distribution of lactase persistence matches distribution of dairy farming (gene-culture ‘coevolution’) (4) Two SNP polymorphisms in LCT gene are associated with lactase persistence, have been selected for BUT takes hundreds, thousands of years for selected SNPs to spread through populations, not yet fixed

41. Natural selection is often geographically-restricted Sickle cell anemia - red blood cell protein polymorphism SS homozygotes - sickle cell disease, early death AS heterozygotes - relatively resistant to malaria AA homozygoes - relatively susceptible to malaria S allele is only favored in malarial area Other red blood cell genes show similar patterns of heterozygote advantage

42. The frequencies of anti-malarial alleles are highest in malarial areas Malaria HB S allele G6PD deficiency allele Here, we see ‘fit’ between alleles and environments, and variation is maintained locally by heterozygote advantage

43. Most-polymorphic loci known in humans are HLA loci, which are involved in immune responses to pathogens Is a positive correlation, among human populations, between HLA heterozygosity levels and virus species richness, suggesting that viruses impose selection for maintenance of genetic variation at immune system loci

44. Worobey et al. (2008), Annual Reviews of Ecology, Evolution and Systematics:

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50. Was severe bottleneck at point of out of Africa, for modern humans Bottleneck, selection due to eruption of supervolcano Toba about 70,000 years ago? Coincide with out of Africa?