.

Introduction to Gene Mapping Techniques

Lecture 2

This class has been edited from several sources. Primarily from Terry Speed’s homepage at Stanford and the Technion course “Introduction to Genetics”. Changes made by Dan Geiger.

Background Readings: Chapter 5 & 6 (190-193) of An introduction to Genetics, Griffiths et al. 2000, Seventh Edition.

2

“Exceptions” to Mendel’s Second Law

Morgan’s fruit fly data (1909): 2,839 flies

Eye color A: red a: purpleWing length B: normal b: vestigial (not fully developed)

AABB x aabb

AaBb x aabb

Observed 1,339 151 154 1,195The pair AB stick together more than

expected from Mendel’s law.

AaBb Aabb aaBb aabb

Expected 710 710 710 710

3

Morgan’s explanation

A A

B B

a a

b b

F1: A a

B b

a a

b b

F2:A a

B b

a a

b b

A a

b b

a a

B b

Crossover has taken place

1,339 151 154 1,195

4

The proportion of recombinants between the two genes (or characters) is called the recombination fraction between these two genes.

It is usually denoted by RF or r or . For Morgan’s traits:

r = (151 + 154)/2839 = 0.107

If r < 1/2: two genes are said to be linked.

If r = 1/2: independent segregation (Mendel’s second law).

5

Recombination Phenomenon(Happens during Meiosis)

Recombination Haplotype

Male or female

:תאי מיןביצית, או זרע

6

כרומוזומים הומולוגיים המראים כיאסמתה

הכיאסמה היא הביטוי הציטולוגי לשחלוף.

Homolog chromosomes showing Chaismata

Chaisma(ta) is the cellular expression of recombination.

Sister chromatids

7

Example: ABO, AK1 on Chromosome 9

Recombination fraction is 12/100 in males and 20/100 in females.

One centi-morgan means one recombination every 100 meiosis.

One centi-morgan corresponds to approx 1M nucleotides (with large variance) depending on location and sex.

2

4

5

1

3

A

A1/A1

O

A2/A2

A

A1/A2

O

A1/A2

A

A2/A2

O OA1 A2

A OA1 A2

A | OA2 | A2

O OA2 A2

Recombinant

Phase inferred

8

גנים4צבע פלפל: אינטראקציה בין

9

Genotype Phenotype

r/r C1/C1 C2/C2 y/y green

R/R C1/C1 C2/C2 Y/Y red

R/R C1/C1 C2/C2 y/y brown

r/r C1/C1 C2/C2 Y/Y yellow

R/R C1/C1 c2/c2 Y/Y orange

r/r c1/c1 c2/c2 Y/Y white

Y : removal of green chlorophyll from fruit y : green chlorophyll in fruit

R : Red carotenoid pigment r : yellow carotenoid pigment

C1; C2 : Two genes with the same function, determine amount of carotenoids .

c1; c2 : Recessive mutations, lower the amount of carotenoids .

גנים4צבע פלפל: אינטראקציה בין

10

הקובעים את הטיפוס האימינולוגי של התא והרקמה. HLA-B ו- HLA-A גנים 2קיימים

A1, A2, A3, A9, A10, A11, A28, A29 אללים שונים: 8 קיימים HLA-Aבגן

B5, B7, B8, B12, B13, B14, B18, B27 אללים שונים: 8 קיימים HLA-Bבגן

כאשר תורמים רקמה, קבלת השתל תלויה בכך שלתורם לא יהיו אללים שאינם נמצאים בנתרם.

אללים אלו יצרו אנטיגנים שבנתרם יגרמו לתגובה אימונית, יצירת נוגדנים, ודחית השתל.

דוגמאות:

גנוטיפ נתרם גנוטיפ תורם תוצאה

A1 A1 B5 B7 A1 A2 B5 B5דחיה

A1 A2 B7 B7 A2 A3 B7 B12דחיה

A2 A2 B7 B7 A1 A2 B5 B7קבלה

A2 A3 B5 B5 A2 A3 B5 B7קבלה

גנים2אינטראקציה בין :אי התאמת רקמות

11

Purpose of human linkage analysis

To obtain a crude chromosomal location of the gene or genes associated with a phenotype of interest, e.g. a genetic disease

or an important quantitative trait.

Examples: Cystic fibrosis (found), Diabetes, Alzheimer, and Blood pressure.

12

Linkage Strategies I

Traditional (from the 1980s or earlier) Linkage analysis on pedigrees Association studies: candidate genes Allele-sharing methods: Affected siblings Animal models: identifying candidate genes Cell – hybrids

Newer (from the 1990s) Focus on special populations (Finland, Hutterites) Haplotype-sharing (many variants)

13

Linkage analysis

14

Fictitious Example for Finding Disease Genes

We use a marker with codominant alleles A1/A2.

We speculate a locus with alleles H (Healthy) / D (affected)

If the expected number of recombinants is low (close to zero), then the speculated locus and the marker are tentatively physically closed.

2

4

5

1

3

H

A1/A1

D

A2/A2

H

A1/A2

D

A1/A2

H

A2/A2

D DA1 A2

H DA1 A2

H | DA2 | A2

D DA2 A2

Recombinant

Phase inferred

15

Association Studies

16

Healthy/Affected versus a bi-allelic Marker (X,x)

H A

X fXH

78

fXA

72

fX

150

x fxH

44

fxA

41

fx

85

fH

122

fA

113

f

235

f

f

f

f

f

fD XHHX

0006.0235

150

235

122

235

78D

So healthy status seems independent of marker X.

17

The Chi-Square test

H A

X fXH

78

fXA

72

fX

150

x fxH

44

fxA

41

fx

85

fH

122

fA

113

f

235

Expected

ExpectedObserved 22 )(

23585

113

)23585

11341(

23585

122

)23585

12244(

235150

113

)235150

11372(

235150

122

)235150

12278( 2222

2

Expected means under assumption of independence of H/A versus X/x.

Using 2 tables, with one degree of freedom, the assertion of independence is not rejected in this example; the probability of 2 is much higher than 0.05.

18

Morgan’s fruit fly data: Can we just be unlucky ?

AABB x aabb

AaBb x aabb

AaBb Aabb aaBb aabbExpected 710 710 710 710Observed 1,339 151 154 1,195

Perhaps genes A and B segregate according to Mendel’s law but we happen to see data that does not support this rule ?What if the answer were:

Fictitious 794 625 625 795

19

The Chi-Square test for Morgan’s data

Expected

ExpectedObserved 22 )(

05.1764710

)7101195(

710

)710154(

710

)710151(

710

)7101339( 22222

Expected means under assumption of independence of the loci A and B.

Use with care; the conversion to probability encodes technical assumptions.

Bb bb

Aa 1339 151 1490

aa 154 1195 1349

1493 1346 2839

This translates to a tiny probability not appearing in the tables; so independence is strongly rejected.

20

Allele-sharing methods

Affected siblings that share an allele more than expected indicate that this allele is near a disease locus. Expected sharing is 1 (out of 2 alleles).

21

Animal/Plant Breeding Methods

Inappropriate for humans. Not practical for large mammals.Not covered in this course, which focuses on computation related to human genetics.

22

בשיטות מעבדתיות מיפוי גנים לכרומוזוםדוגמא: איחוי בין תאי אדם לעכבר

למניעת uvוירוס מסוים, המטופל בקרני-

תאים שונים, וגורם 2פעילותו, נקשר בו זמנית ל-

לממברנות התאים, אחד מהאדם ואחד

מהעכבר, להתאחות.

גרעינים, ולאחר2נוצר תא המכיל

מכן הגרעינים מתאחים ונוצר תא היברידי בו

גרעין המכיל את שני הסטים של הכרומוזומים

(אדם ועכבר).

23

המשך: איחוי בין תאי אדם לעכבר

מסיבות שאינן ברורות, רוב כרומוזומי האדם

נעלמים באופן רנדומלי, וכל תא היברידי מכיל

כרומוזומי 1-4סט שלם של כרומוזומי עכבר, ובין

אדם.

ניתן לעצור העלמות של כרומוזומי אדם

ספציפיים במידה והם מכילים גן המייצר חלבון

העמיד לתנאי התמיסה והאלל של לוקוס גן זה

בצורה זו מזוהה הגן עם אחד הכרומוזומים בעכבר אינו מייצר את החלבון הנדרש.

שנותרו בתא ההיברידי.

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תאים נדרשים ליצר שני אנזימים כדי לגדול )HAT medium(על מצע מסוים

)TK,HGPRT .(TKמיוצר כאשר לוקוס הגן המתאים הינו tk+ -ו .HGPRT

. +hgprtמיוצר כאשר לוקוס הגן המתאים הינו

+tk- / tk- ; hgprt+ / hgprt : נבחר תאי עכבר הומוזיגוטים

-tk+ / tk+ ; hgprt- / hgprt : ונבחר תאי אדם הומוזיגוטים

המשך בשקופית הבאה

כדי שהתאים ההיברידיים יגדלו מתחייב שיישאר לפחות כרומוזום אנושי אחד

.TKובו הגן המקודד את

צביעת הכרומוזומים נותנת דגם פספוס המאפשר זיהוי כל כרומוזומי האדם

הספציפיים שנשארו בכל תא היברידי.

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ניתן להשתמש בהיברידים אלו למפות גנים לכרומוסומים עי שימוש במגוון

של מצעים. יכולת המיפוי תלויה ביכולתנו לזהות תוצר גן המאפשר גידול על

מצע ספציפי (חלבון, אנזים) . למשל כאשר ידוע גן המקודד לחלבון

המאפשר עמידות לתרופה, נבחר עכברים שאינם עמידים ותאים אנושיים

עמידים. Hybrid

Cell line 1 2 3 4 5 6 7 8 A B C D

Human chromosome present gene product

2324

25

- -- -

-++ +

+ +

+

+

: מסקנות

.5: ממוקם לכרומוזום Aגן

.3: ממוקם לכרומוזום Bגן

.1-7: אינו ממוקם לכרומוזומים Cגן

. 1: ממוקם לכרומוזום Dגן

26

Linkage Strategies II

On the horizon (here) Single-nucleotide polymorphism (SNPs) Functional analyses: finding candidate genes

Needed (starting to happen) New multilocus analysis techniques, especially Ways of dealing with large pedigrees Better phenotypes: ones closer to gene products Large collaborations

27

Horses for courses

Each of these strategies has its domain of applicability

Each of them has a different theoretical basis and method of analysis

Which is appropriate for mapping genes for a disease of interest depends on a number of matters, most importantly the disease, and the population from which the sample comes.

28

The disease matters

Definition (phenotype), prevalence, features such as age at onset

Genetics: nature of genes (Penetrance), number of genes, nature of their contributions (additive, interacting), size of effect

Other relevant variables: Sex, obesity, etc.

Genotype-by-environment interactions: Exposure to sun.

29

Example: Age at onset

30

Example: Y-linked disease

31

נורמלי

המופיליה

עיוורון צבעים

המופיליה + עיוורון צבעים

, וקיימת תאחיזה חלקית ביניהם.Xשני הגנים בתאחיזה לכרומוזום

Example: X-linked disease

32

The population matters

History: pattern of growth, immigration

Composition: homogeneous or melting pot, or in between

Mating patterns: family sizes, mate choice

Frequencies of disease-related alleles, and of marker alleles

Ages of disease-related alleles

33

Bottleneck Effects

106 years 105 years

34

Complex traits

Definition vague, but usually thought of as having multiple, possibly interacting loci, with unknown penetrances; and phenocopies.

Affected only methods are widely used. The jury is still out on which, if any will succeed.

Few success stories so far.

Important: heart disease, cancer susceptibility, diabetes, …are all “complex” traits.

We focus more on simple traits where success has been demonstrated very often. About 6-8 percent of human diseases are thought o be simple Mendelian diseases.

35

Design of gene mapping studies

How good are your data implying a genetic component to your trait? Can you estimate the size of the genetic component?

Have you got, or will you eventually have enough of the right sort of data to have a good chance of getting a definitive result?

Power studies.

Simulations.

36

Genotyping

Choice of markers: highly polymorphic preferred.

Heterozygosity and polymorphism information content (PIC) value are measures commonly used.

Reliability of markers important too

Good quality data critical: errors can play a surprisingly large role.

A person is said to be typed if its markers have been genotyped.

37

Preparing genotype data for analysis

Data cleaning is the big issue here.

Need much ancillary data…how good is it?

38

Analysis

A very large range of methods/programs are available.

Effort to understand their theory will pay off in leading to the right choice of analysis tools.

Trying everything is not recommended, but not uncommon.

Many opportunities for innovation.

39

Interpretation of results of analysis

An important issue here is whether you have established linkage. The standards seem to be getting increasingly stringent.

What p-value or LOD should you use?

Dealing with multiple testing, especially in the context of genome scans and the use of multiple models and multiple phenotypes, is one of the big issues.

40

Replication of results

This has recently become a big issue with complex diseases, especially in psychiatry.

Nature Genetics suggested in May 1998 that they will require replication before publishing results mapping complex traits.

Simulations by Suarez et al (1994) show that sample sizes necessary for replication may be substantially greater than that needed for first detection.

41

Topics not mentioned

Exclusion mapping, homozygosity mapping, interference, variance component methods, twin studies, and much more.

Some of these topics plus others are covered in two books:

Handbook of Human Genetic Linkage by J.D. Terwilliger & J. Ott (1994) Johns Hopkins University Press. Ordered, not available at the library.

Analysis of Human Genetic Linkage by J. Ott, 3rd Edition (1999), Johns Hopkins University Press.