1. MCB140 09-15-08 1 “The show so far” 1.There are “particles of inheritance” (units of segregation) … 2.… that pass “unchanged” through generations … 3.… and behave according to certain laws … 4.… and inside the nucleus, which is the part of the cell responsible for heredity, there are chromosomes … 5.… which behave a certain way during meiosis … 6. … and the two sets of behaviors are remarkably concordant … 7.… because the particles (“genes”) actually physically reside on the chromosomes … 8.… as one can prove by the study of eye color inheritance in fruit flies … 9.… and furthermore, a given chromosome carries more than one gene, as discoved when linkage was observed … 10.… and the arrangement of genes on a chromosome is a linear one, with a specific, fixed genetic “distance” separating one gene from another … 11.… that one can experimentally measure by crosses.

2. MCB140 09-15-08 2 This week: what we learn about “the truth” from “exceptions” to the story 1.A particular gene: multiple alleles; complementation criterion for allelism and implications for what a gene is. 2.A particular trait: 1.Gene-gene interactions  expressivity  epistasis 2.Gene-environment interactions  penetrance  norm of reaction 3.Gene and ?  epigenetics

3. MCB140 09-15-08 3 More on what a gene is: allelic series

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10. MCB140 09-15-08 10 Thalassemia (from the Greek word for “the Mediterranean sea”) – a hemoglobinopathy

11. MCB140 09-15-08 11 Two perspectives on the same thing Complementation test: a way to define, what a gene is. (“A gene is a unit of genetic complementation”) If two recessive mutations that exhibit the same phenotype FAIL to complement each other, then they are in the same gene (note that this defines what a “gene” is – it’s the entire entity, recessive mutations in which do not complement each other). If two recessive mutations that exhibit the same phenotype DO complement each other, they are in different genes (by definition). What, exactly, is “complementation”?

12. MCB140 09-15-08 12 Complementation test “Complementation is the production of a wild-type phenotype when two haploid genomes bearing different recessive mutations are united in the same cell.”

13. MCB140 09-15-08 13 The cis-trans test ( aka complementation test) Edward Lewis (NP 1995)

14. MCB140 09-15-08 14 Orgo trans-2-butenecis-2-butene

15. MCB140 09-15-08 15 The cis-trans test, 1949: lozenge (M. Greene) Two different recessive mutants, both with the same phenotype (small eyes and fused facets). What is the relationship of these two mutations to each other? Make two different fly lines and compare their phenotypes. Cis:Trans: lz(BS)lz(g) wt lz(BS) lz(g) wt

16. MCB140 09-15-08 16 lz(BS)lz(g) wt lz(BS) lz(g) wt If flies are normal, then mutations are in different genes. If the phenotype is still mutant, then BS and g must be in the same gene!!! This is a control experiment. The flies will be wild-type regardless of whether BS and g are in the same gene or not. Cis:Trans:

17. MCB140 09-15-08 17 Why this is useful and how this relates to the earlier definition of a gene Mendel lacked the ability to “engineer chromosomes” and perform crosses of such sophistication. Hence, Mendel’s gene is a unit of segregation: the minimal entity that moves through crosses and results in F 2 phenotypes segregating according to Mendel’s laws is a gene. The complementation test offers a crisper definition of what a “gene” is and – as we know from molecular analysis – highlights the molecular complexity of gene architecture.

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20. MCB140 09-15-08 20 A remarkably useful aspect of complementation testing All traits that organisms exhibit – from an ability of convert sugar to ATP (glycolysis, Krebs cycle, oxidative phosphorylation), to the ability to not bleed to death from a simple cut (blood clotting cascade) – require the function of multiple proteins, each encoded by a separate gene. Complementation testing provides a powerful tool to take a large number of organisms exhibiting the same phenotype of interest and “bin” them according to the specific gene that they have a mutation in.

21. MCB140 09-15-08 21 Koopman et al. (1991) Nature 351: 117. XY XX+Sry transgene Sex determination in mammals is a genetic process:

22. MCB140 09-15-08 22 Shmoo Al Capp (1948) – Li’l Abner

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24. MCB140 09-15-08 24 Mating type determination in yeast has both a genetic and an epigenetic component A wild-type haploid yeast cell contains THREE copies of mating type- determining genes: Copy #1: the  1 and  2 genes (silent). Copy #2: the a1 and a2 genes (also silent). Copy #3: An additional copy of genes in item 1, or of the genes in item 2, but active. Whichever genes are contained in copy #3 determines the mating type. This means that a haploid yeast cell – whatever its mating type – has two identical genes in its genome, one of which is active, and the other – silent!

25. MCB140 09-15-08 25 Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22. Rine schematic mate to a cells Fig. 18.14

26. MCB140 09-15-08 26 The data Colonies screened: 675,000 Colonies that mated to a: 295 Major complementation groups: 4 silent information regulators: SIR1, SIR2, SIR3, SIR4 Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.

27. MCB140 09-15-08 27 A trait that results from the action of 3 genes: A  B  C = trait Have two yeast strains: 1 and 2. Let’s MATE 1 and 2 and ask, is this diploid wild- type or mutant. Well, if the mutation in 1 is in gene A, and in 2 – in gene B, then the diploid will look like this: Aa Bb CC = wild-type If the mutation in 1 and in 2 is in gene B, then the diploid will look like this: AA bb CC = mutant

28. MCB140 09-15-08 28 Baur et al. Nature 444: 337. Lagouge et al. Cell 127: 1109. Resveratrol: 2.2 mil Google hits

29. MCB140 09-15-08 29 “Simple” Mendelian inheritance in humans The complexity of the truth

30. MCB140 09-15-08 30 (A)Autosomal dominant; (B)autosomal recessive (C)X-linked recessive (D)X-linked dominant (E)Y-linked.

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32. MCB140 09-15-08 32 Suzanne Vega Solitude Standing Songs in Red and Gray Mitsuko Uchida Mozart Piano Sonatas Note: NOT concerti Tracy Chapman Where You Live

33. MCB140 09-15-08 33 Gene for starting businesses “If you belong to a certain extended family in Seattle, you're probably an entrepreneur. It seems to be about the only career many of the members ever considered. ''It's in our blood'' said Brian Jacobsen, president of Madison Park Greetings, a stationery and gifts company. Mr. Jacobsen's brother, mother, grandfather, two uncles, two cousins and an aunt all started and ran their own companies and say they cannot imagine any other livelihood. Why are so many people in the same clan hooked? Some of them have a theory. They believe that somewhere in their chromosomes lurks an actual entrepreneurial gene -- that their bent for business really is in their blood.” New York Times, Nov. 20, 2003 – p. C8

34. MCB140 09-15-08 34 New York Times, Nov. 20, 2003 – p. C8

35. MCB140 09-15-08 35 The God Gene “Modern science is turning up a possible reason why the religious right is flourishing and secular liberals aren’t: instinct. It turns out that our DNA may predispose humans towards religious faith. … Dean Hamer, a prominent American geneticist, even identifies a particular gene, VMAT2, that he says may be involved. People with one variant of this gene tend to be more spiritual, he found.” N. Kristof, New York Times, 2-12-05

36. MCB140 09-15-08 36 A useful litmus test to distinguish pure conjecture (“handwaving”) from statements that are evidence-based PubMed (via Google). Search: Hamer [au] AND vmat2

37. MCB140 09-15-08 37 Cancelled health insurance? “Kevin McCormick called today. There’s another lawsuit from the Weller family. This time it’s the son of the deceased, Tom Weller. … Apparently, his health insurance got cancelled.” “Because?” “His father has the BNB71 gene for heart disease.” © 2006 Michael Crichton – Next

38. MCB140 09-15-08 38 “Gene Variant Is Linked to Common Type of Stroke” NYT 1/9/07 Japanese researchers have identified a gene variant that appears to predispose a person to strokes, but it seems more prevalent in Asians than in people of European or African descent. In a paper to be published next month in the journal Nature Genetics, researchers write that the presence of the variant raised the risk of cerebral infarction, the most common type of stroke, by 40 percent. Cerebral infarction occurs when blood supply to a part of the brain is obstructed, resulting in death or serious damage to brain cells. The obstruction can be caused by a blood clot, a buildup of fatty deposits in blood vessels or cancerous cells. The researchers studied 1,112 Japanese and found that the variant of the gene PRKCH turned up more often in people who had had strokes. The variant also appeared to be linked to an enzyme, rendering it more active.

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40. MCB140 09-15-08 40 Bruce Springsteen “Cross My Heart” A reminder from the Boss himself Well, you may think the world's black and white And you're dirty or you're clean You better watch out you don't slip Through them spaces in between Born to Run Born in the USA The Rising

41. MCB140 09-15-08 41 Sickle-cell anemia – a brief history “In the western literature, the first description of sickle cell disease was by a Chicago physician, James B. Herrick, who noted in 1910 that a patient of his from the West Indies had an anemia characterized by unusual red cells that were sickle-shaped.” By 1923, it was realized the condition is hereditary. In 1949, Neel realized that patients with SCA are homozygous, and heterozygous carriers have a much milder condition (sickle cell trait).

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43. MCB140 09-15-08 43 Sickle cell anemia NIH: “Sickle cell anemia is the most common inherited blood disorder in the United States, affecting about 72,000 Americans or 1 in 500 African Americans. SCA is characterized by episodes of pain, chronic hemolytic anemia and severe infections, usually beginning in early childhood.”

44. MCB140 09-15-08 44 Pleiotropy Steinberg M. N Engl J Med 1999;340:1021-1030

45. MCB140 09-15-08 45 Linus Pauling, 1949: HbS has different charge!!

46. MCB140 09-15-08 46 V. Ingram, Nature 1956 “On [the existing] evidence alone, it is not possible to decide whether the difference between the proteins, which is in any event small, lies in the amino-acid sequences of the polypeptide chains, or whether it lies in the folding of these chains leading to the masking of some amino-acid side chains.” V. Ingram (1956) Nature 178: 792.

47. MCB140 09-15-08 47 V. Ingram (1956) Nature 178: 792. The third most-famous experiment in the history of molecular biology Digest Hb A and Hb S with trypsin (protease – cuts hemoglobin into ~30 peptides). Separate resulting fragments by electrophoresis, and then by chromatography. Trace the peptide map.

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49. MCB140 09-15-08 49 V. Ingram (1956) Nature 178: 792.

50. MCB140 09-15-08 50 Correct “One can now answer at least partly the question put earlier, and say there there is a difference in the amino-acid sequence in one small part of one of the polypeptide chains. This is particularly interesting in view of the genetic evidence that the formation of hemoglobin S is due to a mutation in a single gene.” V. Ingram (1956) Nature 178: 792.

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52. MCB140 09-15-08 52 RFLP!!!

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54. MCB140 09-15-08 54 Incomplete dominance

55. MCB140 09-15-08 55 Haploinsufficient: 1.Take gene (Q). 2.Q = wild-type. 3.Complete lack of Q (let’s call that allele q) = mutant. Normally, a gene that’s absent makes for a recessive allele (which makes sense – the wild-type copy will still work, so the wild-type allele will be dominant over the “gene is gone” BB King allele). 4.The heterozygote, however (eg Qq), has a mutant phenotype. You have to have two alleles’ worth of protein to have a normal phenotype! “When a gene is haploid, that’s not sufficient” 3.2

56. MCB140 09-15-08 56 Penetrance and expressivity “The terms penetrance and expressivity quantify the modification of the influence on phenotype of a particular genotype by varying environment and genetic background; they measure respectively the percentage of cases in which a particular phenotype is observed when the specific allele of a gene of interest is present and the extent of that phenotype.”

57. MCB140 09-15-08 57 Variable expressivity The importance of genetic background; epistasis

58. MCB140 09-15-08 58 “Treatment Directed at the Relief of Symptoms – Painful Episodes” “In a given year, about 60 percent of patients with sickle cell anemia will have an episode of severe pain. A small minority of patients have severe pain almost constantly. These differences are one manifestation of the heterogeneity of this disease, which complicates the choice of treatment. Episodes of pain are sometimes triggered by infection, extreme temperatures, or physical or emotional stress, but more often they are unprovoked and begin with little warning.” Steinberg M. N Engl J Med 1999;340:1021-1030

59. MCB140 09-15-08 59 Man’s best friend – from a scientific perspective as well

60. MCB140 09-15-08 60 Recessive epistasis (9:3:4)

61. MCB140 09-15-08 61 Dominant epistasis (13:3)

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64. MCB140 09-15-08 64 Complementary gene action (9:7)

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66. MCB140 09-15-08 66 Fig. 3.18

67. MCB140 09-15-08 67 “… the stadium capacity is now officially listed as 75,662”

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69. MCB140 09-15-08 69 Steinberg Curr Opin Hematol 13: 131

70. MCB140 09-15-08 70 “An SCN9A channelopathy causes congenital inability to experience pain” Nature Dec. 14, 2006 “The index case for the present study was a ten- year-old child, well known to the medical service after regularly performing 'street theatre'. He placed knives through his arms and walked on burning coals, but experienced no pain. He died before being seen on his fourteenth birthday, after jumping off a house roof.”

71. MCB140 09-15-08 71 So – let’s think about this The small fraction of African-Americans who are relatively pain-free … … could they be heterozygous for a loss-of- function mutation in SCN9A? In other words, could this be recessive epistasis? If yes, could this suggest that a small-molecule inhibitor of that specific pain receptor could be a more effective analgesic for SCA patients than God-awful parenteral morphine!

72. MCB140 09-15-08 72 Calling Michael Crichton “Gene for …”?! “Patients who are homozygous for the sickle hemoglobin mutation can present with remarkably different clinical courses, varying from death in childhood, to recurrent painful vasoocclusive crises and multiple organ damage in adults, to being relatively well even until old age. Increasing numbers of genetic loci have now been identified that can modulate sickle cell disease phenotype, from nucleotide motifs within the beta-globin gene cluster, to genes located on different chromosomes. With recent success of the human genome project, it is anticipated that many more genetic modifiers of sickle cell disease will be discovered that can lead to the development of more effective therapeutic approaches. The multigenic origin of the variable phenotype in sickle cell disease will serve as a paradigm for the study of variation in phenotypes of all single gene disorders in man.” Curr Opin Pediatr. 2001 Feb;13(1):22-7.

73. MCB140 09-15-08 73 The ob mouse

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75. MCB140 09-15-08 75 Variable penetrance The importance of the environment AND genetic background

76. MCB140 09-15-08 76 60-80%

77. MCB140 09-15-08 77 Hereditary breast cancer caused by mutations in BRCA1 is incompletely penetrant

78. MCB140 09-15-08 78 “Physical exercise and lack of obesity in adolescence were associated with significantly delayed breast cancer onset.” Risk of breast cancer and physical exercise in BRCA1/2 mutation carriers: an example of how the norm of reaction illuminates the modification of a “genetic tendency” by the environment M.-C. King et al. Science 2003

79. MCB140 09-15-08 79 Norm of reaction A plot of carefully measured phenotype in large pool of genetically identical individuals grown under a range of environments.

80. MCB140 09-15-08 80 D. Rio (UCB)

81. MCB140 09-15-08 81 Three mutants that affect facet #

82. MCB140 09-15-08 82 Norm of reaction of different genotypes of the bar locus to temperature.Note, in general, that wild-type flies have a “tendency” to have more facets than the two mutants. WT flies, however, have less facets as the temperature increases, so one cannot claim that the WT genotype predisposes to more facets! Further, ultrabar “tends” to have less facets than infrabar, except at 15 , where it has slightly more facets. This means that the genetic “predisposition” of ultrabar cannot be stated in one sentence.

83. MCB140 09-15-08 83 Achillea millefollium (yarrow)

84. MCB140 09-15-08 84 Take 7 yarrow plants, grow cuttings from each one at different elevations. Measure each “child” at each elevation.

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86. MCB140 09-15-08 86 A fact, and a problem Fact: what we do is a function of what we know (and many other things, of course). Problem: our knowledge comes in shades of gray, but actions tend to be black-and- white.

87. MCB140 09-15-08 87 People with insufficient education in genetics AND statistics and not enough time to look at the primary data 1.Policymakers. 2.Health insurance company officials. 3.Health care providers (i.e., physicians). 4.Journalists who write about science and medicine for major newspapers. 5.The patients themselves. Policy:Data: