7.03 Lectures 35-36 12/10/01, 12/13/01

1

Cancer is:

• among the most common causes of death in the developed world.

breast cancer, lung cancer,leukemias (cancers of white blood cells),

prostate cancer,stomach cancer, etc.

uncontrolled growth is result of multiple mutations accumulating sequentially within a specific cell or cell lineage

• may

• may

Web site for tutorial on DNA sequence analysis relevant to these lectures: web.wi.mit.edu/bio/bio703

7.03 Lectures 35-36 12/10/01, 12/13/01

2

Colon cancer

• second-leading cause of cancer death in U.S. (after lung cancer)

Mutations can occur in either

During past 20 years, many genes have been identified as sites of mutations that predispose to particular cancers. Some genes have been found to be sites of mutations that predispose to cancers of multiple organs. The Rb (retinoblastoma) gene identified by Prof. Weinberg here at MIT in 1986 is involved in both retinoblastoma and osteosarcoma (cancer of bone).

In these lectures we will focus on one particular cancer: Colon cancer

7.03 Lectures 35-36 12/10/01, 12/13/01

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Here is a simplified lineage diagram of human development:

7.03 Lectures 35-36 12/10/01, 12/13/01

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germ line (lineage) =

somatic lineages =

7.03 Lectures 35-36 12/10/01, 12/13/01

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Here we will tally the genetic changes associated with development of colon cancer -- the succession of genotypes

0

1

2

3

4

5

6

- -

+ +

- - - +

- - - +

Step Alleles Alleles Alleles

normal+ + + + + +

+ +

+ + + +

+ +

- - - + - -

7.03 Lectures 35-36 12/10/01, 12/13/01

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Doesn't it seem very unlikely that one cell lineage would, by chance, accumulate all of these mutations?

Two ways to increase the odds of one cell's acquiring this genotype through somatic mutation:

Start with1.

2.

7.03 Lectures 35-36 12/10/01, 12/13/01

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FAP =

elevated risk in first-degree relatives

(via additional mutations, including one affecting second copy of APC gene)

Two heritable germline mutations that predispose to colon cancer:

7.03 Lectures 35-36 12/10/01, 12/13/01

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HNPCC =

elevated risk in first-degree relatives

often before age 50

to any genetic marker had been observed

Two heritable germline mutations that predispose to colon cancer:

7.03 Lectures 35-36 12/10/01, 12/13/01

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What might account for inability to obtain a LOD score >3 for HNPCC?

It turned out that HNPCC showed

Recall our discussion of genetic complexity. In cases where inheritance may be complicated, or where multiple loci may be involved, it is wise to study families of sufficient size that one family alone can yield a LOD score >3.

7.03 Lectures 35-36 12/10/01, 12/13/01

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Genetic linkage analysis of HNPCC

In 1993, linkage studies revealed linkage with LOD > 3 to:

These findings confirmed the suspicion of nonallelic heterogeneity.

Following this strategy, investigators identified several large families in which colon cancer appeared to be segregating according to an autosomal dominant mode.

7.03 Lectures 35-36 12/10/01, 12/13/01

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LOD (Family J) =

LOD (Family C) =

HNPCC and SSR linked:

Pedigrees of families J and C. Numbers above the symbols are patient identifiers; numbers below the symbols indicate age at tumor diagnosis or age at last observation if unaffected; “U” indicates age unknown. If a blood sample was available, the age is underlined. DNA samples from most of the spouses were also available but are not indicated on the pedigrees. Letters signify the site of the tumor: C, colon or rectum; E, endometrium; S, stomach; D, duodenum; O, ovary; P, pancreas; and Br, breast. Marker alleles were omitted to protect confidentiality of family members. General symbols: squares, males; circles, females; all symbols with a diagonal, deceased. Open symbols, no neoplasm detected; solid, colorectal or endometrial carcinoma; dotted, other tumor of HNPCC spectrum; horizontal stripes, colorectal adenoma; and diagonal stripes, diagnosis uncertain.

Science 260: 810-812 (May 1993)

7.03 Lectures 35-36 12/10/01, 12/13/01

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HNPCC and SSR linked:

LOD =

Numbers below symbols indicate age at diagnosis of colon cancer (C) or ovary cancer (O); age at death; or current age, if unaffected.

Nature Genetics 5: 279-282 (November 1993)

7.03 Lectures 35-36 12/10/01, 12/13/01

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Very quickly, within one year, these genetic linkage studies converged with two other lines of research, contributing to the journal Science's decision to declare "DNA Repair" the Molecular Process of the Year in 1994.

7.03 Lectures 35-36 12/10/01, 12/13/01

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The two lines of research which converged with the genetic linkage studies were:

DNA samples from one HNPCC patient

1.

Blood

SSR1

Similar findings were obtained with the same samples with all other SSRs tested, regardless of chromosomal location. This was not an isolated finding. Similar results -- new SSR alleles appearing in tumors -- were observed in many other individuals from other HNPCC families.

7.03 Lectures 35-36 12/10/01, 12/13/01

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While we are accustomed to thinking of someone's

as their

in this case the presence of

is more properly viewed

as a

7.03 Lectures 35-36 12/10/01, 12/13/01

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How to account for new alleles in tumor?

Let's recall what an SSR is and how it is typed using PCR:

7.03 Lectures 35-36 12/10/01, 12/13/01

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Connection to DNA mismatch repair as studied in E. coli and yeast

In humans, it is estimated that

In

that serve to

The second of the two lines of research which converged with thegenetic linkage studies in 1993-1994:

2.

aboutis damaged at

much is known about

7.03 Lectures 35-36 12/10/01, 12/13/01

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A G T C C T T A GT C A G G A A T C

A G T C C T A GT C A G G A T C

A G T C C T A GT C A G G A T C

P (incorporating mismatched base that is not corrected) =

7.03 Lectures 35-36 12/10/01, 12/13/01

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We'll focus here on

In E. coli:

mutants in either of two genes,were identified based on phenotype of

proteins encoded by mutS and mutLtriggering excision of newly synthesized

strand, subsequently

7.03 Lectures 35-36 12/10/01, 12/13/01

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E. coli gene Protein function Yeast homologs Human homologs

SSR instability is increased 103 times in

as compared with wildtype

remarkably like that observed in tumors from HNPCC patients.

Yeast and human genes very similar ("homologous") to mutS and mutL genes were subsequently identified, based on DNA sequence alignments (see web.wi.mit.edu/bio/bio703).

7.03 Lectures 35-36 12/10/01, 12/13/01

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the related genes MSH2 and MLH1 were

respectively.

and not just anywhere on these chromosomes, but to the precise

Eventual conclusions:

1. HNPCC patients in families showing linkage to Chr 2 have:

� complete loss of MSH2 function

7.03 Lectures 35-36 12/10/01, 12/13/01

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2. HNPCC patients in families showing linkage to Chr 3 have:

� complete loss of MLH2 function � mutator phenotype in precancerous growth � rapid accumulation of mutations (not just in SSRs !!!)

3. HNPCC patients in families showing no linkage to Chr2 or Chr3

4. Many sporadic (not obviously familial) colon cancer tumors