MCB140 09-17-07 1

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.”

MCB140 09-17-07 2

“Cancer Free at 33, but Weighing a Mastectomy”

The New York Times, Sunday, Sep. 16, 2007

Deborah Lindner, 33, did intensive research as she considered having a preventive mastectomy after a DNA test.

MCB140 09-17-07 3The New York Times, Sunday, Sep. 16, 2007

MCB140 09-17-07 4www.nytimes.com

MCB140 09-17-07 5

“Little gene cards” – what if it says, BRCA1-mutant?!

“The Lindners share a defective copy of a gene known as BRCA1 (for breast cancer gene 1) that raises their risk of developing breast cancer sometime in their lives to between 60 and 90 percent. Only 30,000 of more than 250,000 American women estimated to carry a mutation in BRCA1 or a related gene, BRCA2, have so far been tested.”

The New York Times, Sunday, Sep. 16, 2007

MCB140 09-17-07 6

“Never ask, for whom the bell tolls”

MCB140 09-17-07 7Hanahan and Weinberg (2000) Cell 100: 57–70.

MCB140 09-17-07 8

Knudsen “two-hit” model

MCB140 09-17-07 9

“Malignancy of somatic cell hybrids” B. Ephrussi et al., Nature (1969) 224:1314 The studies of Ephrussi et al. and Harris provided compelling evidence that the

ability of cells to form a tumor is a recessive trait. They observed that the growth of murine tumor cells in syngeneic animals could be suppressed when the malignant cells were fused to nonmalignant cells, although reversion to tumorigenicity often occurred when the hybrids were propagated for extended periods in culture. The reappearance of malignancy was found to be associated with chromosome losses. Stanbridge and his colleagues studied hybrids made by fusing human tumor cell lines to normal, diploid human fibroblasts. Their analysis confirmed that hybrids retaining both sets of parental chromosomes were suppressed, with tumorigenic variants arising only rarely after chromosome losses in the hybrids. Moreover, it was demonstrated that the loss of specific chromosomes, and not simply chromosome loss in general, correlated with the reversion to tumorigenicity.

The observation that the loss of specific chromosomes was associated with the reversion to malignancy suggested that a single chromosome (and perhaps even a single gene) might be sufficient to suppress tumorigenicity. To directly test this hypothesis, single chromosomes were transferred from normal cells to tumor cells, using the technique of microcell-mediated chromosome transfer. It was found that the transfer of a single chromosome 11 into the HeLa cervical carcinoma cell line suppressed the tumorigenic phenotype of the cells. Many studies have now demonstrated that transfer of even very small chromosome fragments will specifically suppress the tumorigenic properties of certain cancer cell lines.

MCB140 09-17-07 10Ventura et al (T. Jacks) Nature 445: 661

MCB140 09-17-07 11

Beyond Mendel – interactions of gene products in the formation of

traits as revealed by highly modified progeny ratios in

crosses

EPISTASIS (“to stand on top of”)

MCB140 09-17-07 12

Epistasis(or: an epistatic interaction between two loci)1. Pick a trait.2. Find a mutant phenotype #13. Find a different mutant different phenotype (#2)4. Cross the two mutants: get not a mix of phenotypes,

but instead, either phenotype #1 or #2.The term “epistasis” refers to a phenomenon in which an

allele of one gene masks (“stops”) the effects on the phenotype of an allele of a different gene.

The discovery of epistatic interactions between gene products is one of the most powerful tools in genetics – it allows the assembly of individual genes into pathways – and understanding of pathways leads to an understanding of mechanism.

MCB140 09-17-07 13

“For difference determined by one gene…”

MCB140 09-17-07 14Fig. 3.7

MCB140 09-17-07 15Fig. 3.6

MCB140 09-17-07 16

How can one tell, if two organisms under study that

exhibit mutant phenotypes for a particular trait have a mutation in

different genesor in the same gene?

MCB140 09-17-07 17

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.”

MCB140 09-17-07 18

The cis-trans test(aka complementation test)

Edward Lewis (NP 1995)

Are two different recessive mutations that appear to affect the same trait

in the SAME gene or in DIFFERENT genes?

MCB140 09-17-07 19

Orgo

trans-2-butene cis-2-butene

MCB140 09-17-07 20

The cis-trans test, 1949:lozenge (M. Greene)

Two different recessive mutants, both with the same phenotype (small eyes and fused facets).

Are they mutations in the same gene?

Make two different fly lines and compare their phenotypes.

Cis: Trans:

lz(BS) lz(g)

wt wt

lz(BS)

lz(g)wt

wt

MCB140 09-17-07 21

lz(BS) lz(g)

wt wt

lz(BS)

lz(g)wt

wt

If flies are normal, thenmutations 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 whetherBS and g are in the same gene or not.

Cis: Trans:

MCB140 09-17-07 22

MCB140 09-17-07 23Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.

Rine schematic

mate to a cells

Fig. 17.14

MCB140 09-17-07 24

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.

MCB140 09-17-07 25

Baur et al. Nature 444: 337.Lagouge et al. Cell 127: 1109.

MCB140 09-17-07 26

Xeroderma pigmentosum

MCB140 09-17-07 27

What was actually done1. By linkage analysis, it was discovered that the same

disease (XP) can be caused by mutations in 7 distinct loci.

2. The cDNA from each gene was cloned.3. An assay was developed to measure, how sensitive to

UV light cells are.4. Experiment: take cells from patient type A, and

introduce each of the 7 cDNAs, one after another.5. Whichever cDNA restores the wild-type phenotype

corresponds to the gene that is mutated in that cell. Bin XP mutations into “complementation groups”!

MCB140 09-17-07 28

Wait a minute

Ahem.Fine. You take a cell that’s mutant, stick in a gene,

the cell is now wild-type, and you tell us this means the gene you stuck in is the gene that is mutated in the cell.

What if the cell has a mutation in a completely different gene, and the gene you stuck in is just epistatic to the first one?!

Good question. We’ll get to an answer shortly.

MCB140 09-17-07 29http://www.gmi.oeaw.ac.at/

MCB140 09-17-07 30

For complete clarity

1. All genotypes – except in cases of nondisjunction – follow Mendel’s first law, and – except in cases of linked genes < 50 cM away from each other – Mendel’s second law.

2. With the exception of human genetic disease, which is, let’s face it, very rare, and things like blood group inheritance, which belongs mostly on the MCAT (note – its inheritance, not blood groups themselves), the inheritance of phenotype seldom follows Mendel’s laws.

MCB140 09-17-07 31

Complementary gene action (9:7)

MCB140 09-17-07 32

MCB140 09-17-07 33

Recessive epistasis (9:3:4)

MCB140 09-17-07 34

Dominant epistasis (13:3)

MCB140 09-17-07 35

MCB140 09-17-07 36

MCB140 09-17-07 37

MCB140 09-17-07 38

Future e-mail

Professor,Thanks for nothing, BUSTER. First you tell us that deviations from

Mendelian ratios can occur in monohybrid crosses (e.g., in a dominance series), then you tell us they occur in dihybrid crosses and are, in fact, a hallmark of epistasis.

How can one tell the difference?

MCB140 09-17-07 39Fig. 3.18

MCB140 09-17-07 40

“… the stadium capacity is now officially listed as 75,662”

42:12 !!!

MCB140 09-17-07 41

MCB140 09-17-07 42Steinberg Curr Opin Hematol 13: 131

MCB140 09-17-07 43

“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.”

MCB140 09-17-07 44

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!

MCB140 09-17-07 45

Prophylactic bilateral mastectomy (and/or oopherectomy) for BRCA1/2 mutation carriers

“A study of 139 women with deleterious BRCA1 or BRCA2 mutations who were followed at the Rotterdam Family Cancer Clinic. To reduce their risk of breast cancer, 76 of these women chose to undergo prophylactic bilateral mastectomy, whereas the remaining 63 were followed according to a surveillance protocol consisting of a monthly breast self-examination, a semiannual breast examination by a health care professional, and annual mammography. … No breast cancers were observed in the 76 women who underwent prophylactic bilateral mastectomy, whereas eight were detected in the surveillance group. This study … supports the report by Hartmann et al. that prophylactic bilateral mastectomy has an efficacy of at least 90 percent in women classified as at high risk on the basis of a family history of breast cancer. Together [these studies] suggest that of the strategies to reduce the risk of breast cancer in high-risk women, prophylactic bilateral mastectomy is the most effective.Two decades of research have convincingly shown that most women with breast cancer can safely be treated with breast-conserving surgery instead of mastectomy. Thus, it is difficult to accept that prevention should be more extreme than the cure. In this era of molecular medicine, we strive for cancer-prevention options that are more targeted and less invasive than surgical extirpation. Chemoprevention for breast cancer that is as effective and safe as prophylactic bilateral mastectomy is a hope for the future.

Andrea Eisen and Barbara Weber (2001) NEJM 345: 208

MCB140 09-17-07 46

“Physical exercise and lack of obesity in adolescence were associated with significantly delayed breast cancer onset.”

M.-C. King et al. Science 2003

MCB140 09-17-07 47

A Russian proverb that aptly describes most current cancer treatment modalities

«Лучшее средство от кровотечения из носа – жгут на шею».

“The best cure for a nosebleed is a torniquet on the neck.”

MCB140 09-17-07 48

“Chemo” drugs

Intrastrand DNA crosslinking

Apoptosis (programmed cell death)

Depolymerization of microtubules

Cell cycle arrest

MCB140 09-17-07 49Wilson and Elledge (2002) Science 297: 1822.

MCB140 09-17-07 50

Penetrance, expressivity, genetic background, and the environment

“No gene is an island”1. The effect of a given mutation in a given gene

can be modified by that particular individual’s genotype at other loci (SCA). This will lead to variable expressivity – perhaps even incomplete penetrance!

2. Furthermore, the effect of a given mutation in a given gene can be modified by the environment (BRCA) – this will lead to incomplete penetrance (for binary traits, such as cancer), and variable expressivity (heart disease).

MCB140 09-17-07 51

Genetics of continuous variation

MCB140 09-17-07 52Muggsy Bogues, 5’3’’

Yao Ming, 7’6’’

Michael Jordan, 6’6’’

MCB140 09-17-07 53

In a population, phenotypes of individuals for a quantitative trait tend to be normally distributed

MCB140 09-17-07 54

The Great Schism (1901-1935)

“Naturalists”• the origin and

meaning of diversity• populations, groups,

higher taxa• gradualism• ultimate causation

“The Mendelians”• transformation of

genes• individual genes/loci• saltationism• no “why” questions

“Only the experimental method would permit an objective discussion of the theory of evolution, in striking contrast to the older speculative method of treating evolution as a problem of history.” (T.H. Morgan)

MCB140 09-17-07 55

Synthesis: Population Genetics

• H. Nilsson-Ehle, R.A. Fisher, J.B.S. Haldane, S. Wright: continuous phenotypic variation is not at odds with particulate inheritance:

multiple loci + epistasis• S.S. Chetverikov: naturally occurring

recessives as food for natural selection.

MCB140 09-17-07 56

Morgan, ch. 8

MCB140 09-17-07 57

Hermann Nilsson-Ehle

MCB140 09-17-07 58

Central limit theorem Carl Friedrich Gauss

If a variable is the sum of many independent variables, then its distribution will be normal:

e x 2

MCB140 09-17-07 59Fig. 3.17

MCB140 09-17-07 60

“Additive effects of genes”

Fig. 3.22

MCB140 09-17-07 61

MCB140 09-17-07 62

Talking to laypeople(and biochemistry majors)

1. Technology and science have advanced to the point where “DNA research” is materially affecting public health – both at an individual level, and at a societal one.

2. Our ability to interpret genetic data lags behind our ability to act intelligently on those data.

3. While this gap is being worked on, and with the critical exceptions of carrier (or prenatal) testing for inherited human disease such as Tay-Sachs, and cystic fibrosis, and also of breast, colon, thyroid, stomach, and skin cancer for individuals with strong family histories of the above – “the DNA stuff” is best left alone (for now) – from an action perspective – but not from that of knowledge!