2000MSC Molecular Genetics

Lecture Notes
2015

Undergraduate Course, School of Medical Science, Griffith

University, Gold Coast campus, Queensland, Australia.

TOPIC 1 MENDELIAN GENETICS

1.0 INTRODUCTION
Genetics is the study of heredity. Three main branches:
1. Transmission genetics: the study of transmission of traits from
generation to generation. Pioneered by Gregor Mendel in 1865
2.

Molecular Genetics: the study of the composition and role of

genetic material based on the central dogma that DNA is the

template for RNA which encodes proteins. This explains the
transmission of traits first described by Mendel.
3.

Population Genetics: the study of variation and evolution in

populations. We know that DNA accumulates changes (mutations)

which result in phenotypic variation between individuals in a
population.

Natural selection can then act on that variation and

the population will change over time, ie the population will evolve.

WE

SHOULD ALWAYS KEEP IN MIND THE RELATIONSHIP

BETWEEN THE THREE BRANCHES

1.1 MENDELS EXPERIMENTS

(Refer text chapter 3, pages 43-55)

Mendel 1856

Crossed pea plants easy to grow, crossbred artificially, grows

to maturity in one season

Used 7 visible features (traits), each with 2 visible alternate

forms (phenotypes), used pure-breeding strains

[Please revise how chromosomes move in meiosis to produce

gametes: try youtube.com/watch?v=_IzfJSxa-uA]

1.1.1 MENDELS OBSERVATIONS

OF

PLANTS

WITH

ONE TRAIT

The Monohybrid Cross

Crossing two pure-breeding plants resulted in one phenotype in

the F1 progeny

Eg. plant height tall

short

tall

Inbreeding F1 progeny (self fertilisation) resulted in two

phenotypes in the F2 generation

The phenotype seen in the F1 progeny was 3 times more

common in the F2 progeny than the other phenotype

3:1 phenotypic ratio; 1:2:1 genotypic ratio

This led to Mendels first 3 postulates

1.1.2 MENDELS FIRST 3 POSTULATES

1. Unit factors exist in pairs (unit factors control genetic
characters/traits)

Prior to this, thought that one per trait with parental traits
blending in offspring

Factors separate into germ cells (observed traits of seeds;

round/wrinkled, green/yellow etc)

Fertilisation results in individuals with one factor from each

parent

2. Dominance/Recessiveness

In the pair of unit factors for a single trait in an individual, if

each is unlike the other, one unit factor is dominant and the

other is recessive
3. Law of Segregation:

In the formation of gametes, the two factors for any

trait separate (segregate) randomly, so that each ends
up in a different gamete.

Separation of factors gives gametes with equal chance

of each factor due to random union of gametes

1.1.3 MENDELS OBSERVATIONS

OF

PLANTS

WITH

TWO TRAITS

The Dihybrid Cross

To see if the law of segregation applied to more than one trait,

Mendel crossed plants that were purebreeding (dominant and
recessive) for two traits eg. Seed colour and shape

He found:
o F1 one phenotype of each separate trait
o F2 9:3:3:1 phenotypic ratio

F2 ratio due to combination of two independent 3:1 ratios

This may be done with a Punnett square to allow the genotypes and
phenotypes from a cross to be visualised easily (Reg. Punnett
Cambridge, founded Journal of Genetics 1910, developed Punnet
swquare 1905, discovered linkage 1906).

9 different genotypes, 4 phenotypes in a 9:3:3:1 ratio

This led to Mendels second law: The Law of Independent

Assortment

MEIO1.1.4

LAW

OF

INDEPENDENT ASSORTMENT

Segregation of two factors for a trait are independent of the

segregation of other factor pairs in the formation of gametes

Individual

members

of

pairs

of

alleles

segregate

independently of one another during gamete formation

Eg.

AaBb
Where

and

alleles

separate

independently
of B and b alleles

AB

Ab

aB

ab

This gives 4 possible types of gametes, therefore if you cross 4

gametes

with

another

gametes

you

get

16

possible

combinations
CONCLUSIONS

Mendel determined laws that demonstrate inheritance of traits

Law of segregation: two factors per cell for a particular

trait separate independently into gametes

Law of independent assortment: separation of pairs of

alleles are independent of each other

Mendel had no knowledge of chromosomes or genes

Used maths and probabilities to determine laws

Also persistence, repetition to verify results and logic to reach

conclusions

We can correlate Mendel's Postulates with the behaviour of

chromosomes during meiosis

Were his results too good?

Luck did not use linked genes or incomplete dominance

1936 Fisher evaluated Mendels data to determine if Mendel

was neutral or objective in his studies or had his conclusions in
mind

Found that results were closer to expected frequencies than

should have been

Looking at whole series of experiments, there was a 1/14000

chance of getting results as Mendel had claimed

However, others repeated, giving ratios similar if not better than

Mendels

Unconscious bias? Experience modifying expectations? Some

subjective interpretation? Fisher was incorrect? Medels paper
abbreviated?

1.2 TERMINOLOGY
TERMINOLOGY I

Genes: Mendels hereditary factors; segment of DNA specifying

RNA or polypeptide

Alleles: alternate forms of a gene, normal = wild-type

o NB: Alleles are not restricted to two, there may be many
alleles for a gene

Genotype: genetic constitution

Phenotype: characteristics

that result

from genotype and

environment
o Identical twins: same genes, therefore different phenotype
from environment

Locus: position of gene on chromosome

Diploid: two copies of a gene (two sets of chromosomes)

Haploid: one copy of a gene (one set of chromosomes)

Homologous chromosomes: same genes, may be different alleles

Homozygote: genotype with same allele (eg. AA)

Heterozygote: genotype with different alleles (eg. Aa)

TERMINOLOGY II

Pure-breeding: parent that produces only one progeny like

themselves

F1: first filial generation

F2: formed by crossing with F1

Testcross: cross of hybrid F1 with homozygous recessive to

determine unknown genotype

Backcross: cross of hybrid F1 with parent

o NB: testcross is a type of backcross

Monohybrid: an organism who is heterozygous at one gene locus

Aa

Dihybrid: an organism who is heterozygous at two gene loci

AaBb

CONVENTIONS

Genes on different chromosomes: AaBbCcDd

Linked genes:

ABCD
abcd

Indicating haplotypes or arrangement of linked alleles

Capital letter for dominant genes

Small letter for recessive genes

1.3 TESTCROSSES
A testcross may be used to detect the genotype of an individual
who expresses a dominant trait. Eg. How would you determine if a
tall plant is DD or Dd?
For a testcross, cross with homozygous recessive to discover
genotype of unknown
1.3.1 TESTCROSS WITH ONE TRAIT
10

Results give unknown genotype

1.3.2 TESTCROSS WITH TWO TRAITS

In this case, it may be used to detect the genotype of an individual
who expresses two dominant traits
AABB

aabb

(AB)

(ab) gametes

AaBb

aabb

(AB)(Ab)(aB)(ab)

(ab) gametes

AaBb
1

Aabb
1

aaBb
:

aabb
1

11

4 phenotypes, 4 genotypes, in equal ratios when the unknown is a

dyhybrid ie AaBb
If unknown is AABB, then 1 phenotype and 1 geneotype ie AaBb
[Do the Punnet square]

1.4 THE FORKED-LINE

METHOD FOR

F1 X F1 CROSSES

Consider that each gene pair moves separately during gamete

formation

In Aa x Aa cross (F1 generation) we know that will have yellow

and will have green seeds and in Bb x Bb cross we know that
will have smooth and will have wrinkled seeds

The product law is applied ie. when 2 independent events occur

simultaneously, the probability of the two outcomes occurring in
combination is equal to the product of their individual
probabilities of occurrence

12

The forked line method can also be used for a trihybrid crosses.
Study text figure 3.9

1.5 EXTENSIONS

OF

MENDELISM

(Refer text chapter 4, pages 74-86)

Extensions of Mendelian analysis include:

o that a gene can exist in many different allelic states
o that a particular gene can affect several different traits
o that a particular trait can be affected by several different
genes
o genetic and environmental factors influence phenotypic
variation

COMPLETE DOMINANCE

Phenotype of homozygote same as that of a heterozygote

o Eg. PKU (Phenylketonuria)
o overproduction of phenylpyruvic acid (in PKU patients)

13

due to inactive phenylalanine hydroxylase

o Build up of phenylalanine leads to mental retardation
o Due to alleles of enzyme phenylalanine hydroxylase
o Homozygote dominant: Phy Phy (PP) unaffected
o Heterozygote: Phy phy (Pp) unaffected
o Homozygote recessive: phy phy (pp) - affected

PARTIAL (INCOMPLETE) DOMINANCE

Heterozygote shows intermediate phenotype

Eg. Snapdragons

New phenotype seen in heterozygote

Not seen in Mendels results

CO-DOMINANCE

Both phenotypes in heterozygote

Eg. ABO human blood groups

Due to enzyme that adds sugar to blood group antigens on RBC

A N-acetyl sugar

B Unacetylated sugar

O No sugar

Alleles: IA, IB, i

14

Blood groups: A, B, AB, O

Genotypes: IAIA, IAi; IBIB, IBi ; IAIB, ii

IAIB AB individuals produce A and B antigens

Epistasis

Gene interactions

Phenotype of one gene influenced by genotype of one or more

other genes

Results in variations from simple Mendelian ratios

May be dominant or recessive epistasis, different ratios for each

Interaction of genes changes simple Mendelian ratios

Eg.

Pigment in violets, eg of recessive epistasis where dd is

epistatic to G and g
Pink

D active enzyme
d inactive enzyme

Red

D enzyme g pink
G

D enzyme G

white
White
DDGG

DdGg

ddgg

DdGg

D_G_ : D_gg : dd_ _

9

: 4

15

Penetrance

Incomplete penetrance: some individuals with a particular

genotype do not always express the associated phenotype

Complete penetrance: genotype always expressed in associated

phenotype = 100% penetrance, incomplete penetrance is when
the genotype is expressed in the associated phenotype <100% of
the time

Variable Expressivity

Genes expressed to different degrees in different individuals

Human disease, severe to mildly affected with same disease

gene

Eg. Polydactyly

Pleiotropy

Multiple phenotypic effects from single defective gene

Eg 1. Porphyria variegate: build-up of porphyrins in the brain

due to inadequate metabolism of haemoglobin porphyrins

Features include abdominal pain, fever, muscular weakness,

racing pulse, insomnia, headaches, vision problems, delirium,
convulsions, photosensitivity

Eg. 2. Marfan syndrome Fibrillin-1 mutation that affects many

organs

Polygenic Traits

More than one gene contributes to one phenotypic trait eg. eye

16

colour

Often environmental influence eg. human height, weight, skin

colour

Quantitative traits are polygenic

Lethal Alleles

May be recessive or dominant

Eg. Huntingtons disease

May change simple Mendelian ratios

1.6 QUANTITATIVE INHERITANCE

Continuously varying traits such as height in humans are

influenced by a large number of genes, each segregating
different alleles; termed polygenic

These traits are also often influenced by a host of environmental

factors, each having a small effect

This combination of genetic and environmental factors often

creates a wide range of phenotypes, described in quantitative
terms ie. Quantitative inheritance

e.g. height in humans, genetic + environment such as nutrition

1.7 PEDIGREE ANALYSIS

Can deduce modes of inheritance and genotypes of individuals

by looking at pedigrees

Standard conventions to represent pedigrees

17

1.7.1 MODES

OF

INHERITANCE

Autosomal not on sex chromosomes

Y-linked very rare

Sex-linked generally refers to X-chromosome

Also note variations within the normal categories due to

epistasis,

variable

expressivity,

incomplete

penetrance,

pleiotrophy

Autosomal Dominant

At least one parent affected

18

Matings usually give half affected offspring

In general:

Has vertical pedigree pattern

Less severe disorders than recessive

Often show variable expressivity

May show anticipation, ie. earlier onset, more severity with

each successive generation

Examples:
o Brachydacttly (short fingers)
o Polydactyly
o Achondroplastic dwarfism
o Huntingtons disease

Autosomal Recessive

Parents not usually affected

Cousin marriages more common in parents of affected

Inherited from both parents

In general:

Less variable expressivity since two copies of allele needed to

see phenotype

Has horizontal pedigree pattern

Examples:
o PKU
o Albinism
o Cystic fibrosis
o Sickle-cell anaemia

19

TOPIC 2 LINKED AND SEX LINKED GENES

2.1

CELL CYCLES

(Review text chapter 2)

2.1.1 PROKARYOTES

Cell cycle involves DNA replication with cell division by

binary fission immediately after

No special structures for division, no DNA condensation

Single circular DNA molecule replicated

Attached to membrane cell wall divides

2.1.2 EUKARYOTES

Nucleated cells

Mitosis & meiosis

Most cells/organisms are diploid (2n)

Gametes are haploid (n)

Diploid ----------> Haploid ----------> Diploid

20

meiosis

fertilisation

Ploidy = number of chromosome sets

Interphase = phase between cycles (cells most active time)

Mitosis can occur in cells of any ploidy

Meiosis can only occur in even ploidy cells

Meiosis needs homologous chromosomes

Meiosis results in VARIATION of offspring

o

Meiosis I = homologous chromosomes separate

Meiosis II = chromatids separate

2.1.3 CONSEQUENCES SEXUAL REPRODUCTION

Meiosis

Halves ploidy

Independently assorts chromosomes

Allows recombination between linked genes

Fertilisation

Random union of gametes from parents

Variation in chromosome combinations & gene combinations

on chromosomes in the offspring

Mendels' Laws
1. Segregation - one allele in any gamete
2. Independent Assortment - segregation of alleles, independent
of allele segregation for other genes

21

2.2

Genes On Different Chromosomes

Show independent assortment, segregation

Providing the following

1. Not linked
2. Complete dominance
3. No lethal genes
4. No epistasis

TESTCROSS F1
AaBb

aabb

(AB)(Ab)(aB)(ab)

(ab)

AaBb : Aabb : aaBb :aabb

1

: 1

22

DIHYBRID CROSS
F1*F1

OF

F1

AaBb x AaBb

A_B_: A_bb : aaB_ : aabb

9

2.3

3 : 1

LINKED GENES

(Refer text page 106-9)

Linkage

Linkage:

variation

in

gametes

due

to

different

combinations of linked genes

Crossing over occurs when homologous chromosomes align

during Prophase I, pieces of chromosomes may be exchanged

Proposed by Thomas Morgan: fruit flies, 1908 Columbia

University, New York, found genetic crossing over

Chiasmata discovered in 1909 by Jansessns, Jesuit priest and

Professor, a physical explanation for crossing over

Chiasmata are points

of

chromosomes during

Prophase I

Linked

genes on the same chromosome

Do NOT show independent assortment of alleles

Tend to be inherited together

Genes very close - little crossing-over

genes

are

overlap

on

homologous

23

Genes further apart - more crossing-over

1% crossing-over = 1cM (centimorgan)

1cM = chromosome map unit

Crossing-over = recombination

Maximum Detectable = 50%

2.3.1 DETECTION OF LINKAGE

Detected
ie.

by

deviation

Variation

from expected ratios

from

1:1:1:1

testcross

&

9:3:3:1

dihybrid F2
Eg.

TESTCROSS
AB

ab

AB

ab

F1

AB

ab

ab

ab

AB

ab

Ab

aB

ab

ab

ab

ab

24

[------------] = crossing over (nonparentals)

If genes are 20cM apart, then:

recombinants = 20%

parentals = 80%

Recombination due to crossing-over

Phenotypes different to original parents, therefore sometimes

called non-parentals

Non-Recombinants = no crossing-over

Phenotypes same as original parents, are called parentals

Eg. If genes 10cM apart

o

= 10% recombinants (5% for each type)

& 90% non-recombinants (45% for each type)

Proportions Testcross
% recom.

Parental

Non-

Parental
10cM

0.45

0.45

0.05

0.05

20cM

0.40

0.40

0.10

0.10

50cM

0.25

0.25

0.25

0.25

Not Linked

1.00

1.00

1.00

1.00

2.3.2 Determining Distance

Use testcross to determine distance between linked genes

25

BD

F1

bd

BD

bd

(BD)

(bd) gametes

BD

bd

bd

bd

(BD)(Bd)(bD)(bd)

(bd) gametes

BD
bd
Nos

bd

176

% recombination

bd

Bd

bd

bd

190

16

bD
18

= 16+18
total
= 8.5cM

2.3.3 LINKAGE MAPS

Amount

of

crossing-over

used

to

determine

distances

between genes

Termed Linkage Maps

Distance is additive

4cM

5cM

A---------------------B-------------------------------C
add distance A - C = 9cM

26

Genome Maps in various organisms use linkage data

& somatic cell hybridization & in-situ hybridization

Data to combine results to give maps of various chromosomes

2.4

GENE MAPPING

Three Methods
i)

Somatic Cell Hybridization

Follows segregation of gene product with a particular

chromosome. Text P 123

ii)

In-Situ Hybridization
Bind cloned gene probe to chromosome directly

iii)

Linkage Studies

Probability that particular pedigree/s with 2 traits reflects

linkage between them.

Can be detected on autosomes and sex chromosomes

Calculate probability that 2 traits independently assort

according to non-linkage.

Then calculate probability that same data results from linked

genes.

Calculate ratio of these probabilities and convert to logarithm

This generates LOD scores (LOD = Log of the odds for

linkage)

LOD SCORES

Linkage between markers detected by computer analysis of

gene ratios

IF:

27

Lod score of 3 = 1000:1 odds for linkage

Lod score of -2 = 100:1 odds against linkage

Lod scores can be added over families

2.5

SEX-LINKED INHERITANCE

(Refer text chapter 4 pages 88-91)

Sex can be used as a marker for a disorder

Genes located on the sex chromosomes show sex-linked

inheritance
o

Refers to genes in the differential segment of X or Y

Genes on X are called - X-linked

Genes on Y are called - Y-linked (holandric)

Mostly X-Linked

Mode of inheritance will indicate X-chromosomal localisation

of the gene in question

ie. No male to male transmission in X-linked & obligate male

to female transmission in X-linked

Since X-chromosome is much larger & therefore has much

larger differential segment, there are many more X-linked
disorders (X-linked disorders almost always recessive)

First documented by Thomas Morgan 1910

Males hemizygous for for X-linked genes

2.5.1 X-LINKED RECESSIVE

Tends to skip generations

More affected males than females (affected female requires

28

mother & father affected which is rare)

Half sons of carrier females are affected

Lethal only in males

Examples:
o

Haemophilia A & B

Duchenne Muscular Dystrophy

Red-Green Colour Blindness

2.5.2 X-LINKED DOMINANT

At least one parent affected

Affects males and females equally

Affected female transmits to half sons

Affected male transmits to none of his sons but ALL

daughters

In general, males have more severe & less variable forms of

the disease

Examples:
o

Vitamin-D resistant rickets

Goltz's syndrome, usually lethal in males, multiple

abnormalities

2.5.3 SEX-LIMITED

AND

SEX-INFLUENCED INHERITANCE

Genes not on sex chromosome

Sex plays a role in expression of a phenotype

SEX-LIMITED CHARACTERISTICS

In Sex-limited inheritance, expression limited to one sex eg.

Plumage in domestic fowl, milk production in female dairy
cattle
29

SEX INFLUENCED CHARACTERISTICS

In sex-influenced inheritance, sex influences expression

o

May be hormonal influence

Eg. Pattern baldness in humans (also called premature

<35yrs baldness)
o

Rare in females

Related to testosterone levels

Bald allele HB, normal allele HN

HB - dominant males, recessive females

Normal
Males:

HNHN

Females

HNHB

Bald
HNHB,HBHB
HBHB

HNHN

2.6

SEX CHROMOSOMES

(Refer text chapter 7 pages 178-188)

2.6.1 CHROMOSOMAL BASIS OF SEX DETERMINATION

HUMANS (XY)

2n = 46

= 44 autosomes + XX or XY

Gametes (male) = 22A + X or 22A + Y

(heterogametic)

Gametes (female) = 22A + X

(homogametic)

30

MOST INSECTS

Protenor method of sex determination (XX-XO)

o

Male, 2n = 13

12A + X

Female, 2n = 14 12A + XX

Lygaeus method of sex determination (XX-XY)

*NB.

Male, 2n = 14

12A + XY

Female, 2n = 14 12A + XX

MALES NOT ALWAYS THE HETEROGAMETIC SEX

Eg. Some birds, butterflies, fish, reptiles, chickens (ZZ/ZW)

Females exhibit either Protenor

or Lygaeus method of sex

determination

Use notation ZZ (male)/ZW (Female) where ZW is female

heterogametic

2.6.2 DISJUNCTION

During Anaphase I of meiosis the sex chromosomes separate called dysjunction

X & Y chromosomes have short homologous pairing segment

This allows sex chromosomes to pair at metaphase. They then

separate at Anaphase I

Results in male gametes : 22A + X or 22A + Y

In females, XX pair homologously & separate at Anaphase I

giving : 22A + X gametes

2.6.3 NON-DISJUNCTION OF SEX-CHROMOSOMES

31

In females : occasionally the X-chromosome do not separate

at Anaphase I of meiosis

Results in two abnormal gametes:

22A + XX

22A + O

Fertilisation can then result in:

i) 44A + Y

- lethal

ii) 44A + X

- Turners' Syndrome

iii) 44A + XXY

- Klinefelters' syndrome

iv) 44A + XXX

- Triple X female

Male

Female
22A + XX
44 + XXY

22A +

22A
44 + Y

Y
22A +

(lethal)
44 + X

(Klinefelters)
44 + XXX

(Turner)

(Triple X)

2.6.4 HUMAN SEX CHROMOSOMAL ABNORMALITIES

TURNER SYNDROME (X)

REFERRED TO AS

(XO)

Sterile, female phenotype

Undeveloped breasts, rare menstruation

Webbing of the neck

Short stature

Normal Intelligence

KLINEFELTER SYNDROME (XXY)

32

Male phenotype

Varies normal phenotype --> sterile male with breast

development

Broad pelvis, breast enlargement

Generally reduced intelligence but not always

TRIPLE X FEMALE (XXX)

Normal female phenotype, including fertility, maybe taller,

majority never detected, 1/1000 births.

Non-Dysjunction can also Occur in Males

Non-dysjunction at Anaphase I can lead to

o

44 + XXY

or

(Klinefelters)

44 + XO
(Turners)

Non-dysjunction at Anaphase II can lead to

o

22XX or 22YY

+ normal 22A + X

44 + XXX or

(Triple X)

44 + XYY

(XYY male)

XYY MALE

Male phenotype, normal IQ

Tall due to increase SHOX genes, 7cm on average, may be

fertile

A fascinating example of bad data and instant social

stigmatization based on genetic testing. So called XYY super
male criminal now considered false.

33

2.6.5 SEX DETERMINATION

sex

is

determined

by

IN

the

MAMMALS
dominant

effect

of

the

chromosome

presence of Y = male; absence of Y = female

Maleness is a result of:`

Y chromosome loci that cause differentiation of embryonic

gonads to form testis

TDF (Testis Determining Factor) is a gene on the Y

chromosome in a regions called SRY (Sex-determining
Region-Y) which is just outside the pseudo-autosomal region

SRY was found by studying individuals whose sex was

inconsistent with their chromosome constitution eg XX males
and XY females.

XX males carry a small piece of the Y chromosome in one to

their X chromosomes

XY females have a small region of their Y chromosome

deleted

Y chromosome has a small pseudoautosomal region that

has genes found on X

2.6.6 ANDROGEN INSENSITIVITY SYNDROME

Aka. testicular feminisation synfrome

46, XY - normal karyotype but sterile female phenotype

Normal male hormone (testosterone) levels, but testes

34

develop internally

Male sex characteristics do not develop

Due to defective X chromosome gene for androgen receptor

protein which is activated by testosterone to develop male
phenotype, therefore testosterone fails to act & female sex
characteristics develop

Androgen receptor protein

o

Coded for by AR gene on X, defective gene Tfm

(testicular feminisation mutant)

Therefore XY females can respond to testosterone if the

hormone is administered, resulting in male characteristics

2.6.7

THE SEX RATIO

Femal

Male
X
Y
XX
X

XX

Y
X

Expect

1:1 male:female

Actually

Male

conception <1.2 :

birth

20 yrs

85 yrs

0.62

Reasons: greater mobility of smaller Y-bearing sperm?

More male deaths in utero & later, - major reason, masking of

1.05

Female

unfavourable X-genes in females, not males

35

2.6.8 X-INACTIVATION & DOSAGE COMPENSATION IN

FEMALES

Since females have two chromosomes, why don't they

produce twice as many X chromosome gene products
compared to males?
o

Answer: X-Inactivation (Lyon Hypothesis) results in

dosage compensation

In females, interphase somatic cells, one X condenses into

small dark object = Barr Body (named after discoverer
Murray Barr)

A Barr body is composed of 'inactive' condensed chromatin,

therefore female & male, X gene products are equal
o

Number Of Barr Bodies = No. Of X Chromosomes - 1

Eg. XXX female has 2 Barr bodies

Used as sex test eg athletics

In

mammals,

X-inactivation

occurs

during

early

embryogenesis when the embryo consists of just a few

thousand cells

Each cell makes an independent decision to randomly silence

one of its X Chrs

This X Chr remains inactivated in all the descendants of that

cell leading to clones of cells with different activated X
chromosomes

In cells that become oocytes, X inactivation is reversed,

therefore cells entering meiosis have 2 active X chromosomes

Thus female mammals contain 2 types of cell clones, some

with the maternally inherited X inactivated, others with the
paternal

A female that is heterozygous for an X-linked gene is

therefore able to show 2 different phenotypes

Eg.

Fur

colouration

in

cats

and

mice

are

X-linked
36

(heterozygote = tortoiseshell)

TORTOISESHELL CATS

Gene for colouring in cats on X chromosome

Why do Turner Syndrome (XO) patients show phenotypical

abnormalities when XX female cells inactivate one X chromosome
normally?

Answer: A small number of genes on the Barr body are still active
and are thought to be essential in a double dose for proper growth
and development. Additionally both Xs are required (Barr body
becomes unmethylated/active again) for normal ovarian function.

37

TOPIC 3 MEDICAL GENETICS

4.1 CHROMOSOME STRUCTURE

4.1.1 Chromosomes

Composed of DNA core with various proteins

DNA & proteins is termed chromatin

Sequence of bases in DNA determine types of genes

Genes lie in linear fashion along chromosomes

4.1.2 STRUCTURE
(Refer text figure 12.9)

Chromatin is DNA coiled around histone proteins (which are +ve

charged)

8 histones complex forms nucleosomes

147 bp of DNA coiled twice around histone core containing:

-

2xH2A, 2xH2B, 2xH3, & 2xH4

H1 associated with DNA that links nucleosomes

Nucleosomes are then coiled with 6-7 nucleosomes per turn

Further packing & coiling prior to cell division associated with

non-histone proteins

DNA must be packed to fit into cells, also to allow movement

1 human diploid cell = 6.4 picograms DNA

~ 2 metres length

This fits onto a nucleus with a size of 5-10 m!

38

4.1.3 Chromatin Remodeling

Chromatin remodeling must occur to allow the DNA to be

accessed by DNA binding proteins for gene expression and
replication.

Histones play a role in chromatin remodeling

Histone tails may be acetylated or methylated; related to

increased gene activity

BUT methylation of bases TURNS OFF genes

Involved in normal development eg. X inactivation

We call this EPIGENETIC modification ie. changes in gene

expression not related to DNA sequence

4.1.4 Chromosome Morphology

At metaphase most tightly coiled to allow movement

Inactive in terms of gene expression

Each chromosome has 1 molecule of DNA - different length &

type of DNA sequences

= different genes on each

chromosome

BUT homologous chromosomes have same genes (may have

different alleles)

Prior

to

cell

division,

chromatids joined at the

chromosomes

have

centromere

Centromeres mediate chromosomal migration during mitosis

and meiosis, along with

metaphase

kinetochore

Centromeres contain much repetitive sequence in higher

eukaryotes

All chromosomes have a short arm and a long arm separated by

the centromere
o Short arm is designated p (petit)
39

o Long arm is designated q (queue)

Tips are referred to as telomeres

Telomere DNA sequences consist of short tandem repeats

involved in stability and

o Otherwise

integrity of the chromosome.

chromosome

ends

would

resemble

DS

breaks and become degraded by nucleases or fuse to

other chromosomes

Telomerase prevents telomere shortening

Numbering from centromere to telomere: cen ter, based on

bands of different staining

Chromosome forms: Metacentric, submetacentric, acrocentric,

telocentric

Abnormal chromosome types:

o Acentric, no centromere (generally lost)
o Dicentric, 2 centromeres

4.1.5 Chromosome Banding

Centromere position and arm ratios can be identical in different

chromosomes

Therefore limited ability to identify all chromosome pairs

Dyes can be used to produce reproducible patterns of bands on

chromosomes

Chromosome banding is a standard and indispensable tool for

cytogenetic analysis

G, Q, R and C banding, chromosome painting

Each chromosome has a unique bar code

Obtain chromosomes from blood etc

Culture and stimulate cell division

Arrest in metaphase, colchicine may be used

40

4.1.5 Karyotyping

Representation of chromosomes within a cell

Chromosomes paired and in order of size

Can be used to study chromosome number and morphology

Normal karyotype: 22 pairs autosomes (numbered 1-22) and a

pair of sex chromosomes (XX or XY)

Chromosomes identified based on size, morphology and banding

patterns

Karyotypes are presented in standard form

The total number of chromosomes followed by a comma and

then the sex chromosome constitution

Can be followed by a short hand version of any abnormalities

Eg. female dog with increased length of the short (p) arm of
chromosome 2: 78, XX, 2p+

4.2 PLOIDY

Haploid - one set of chromosomes (n)

Diploid - 2 sets (2n)

4.2.1

EUPLOIDY

(Refer text chapter 8)

Multiples of complete haploid sets, includes

o Diploid
o Polyploid - more than 2 haploid sets

Common in crop plants: bananas, cotton, peas,

coffee

Often gives cells larger size, therefore larger, more

41

vigorous plants

Vertebrates rare, occasionally in invertebrates

Need even no. polyploidy in nature : 4n ---> 2n

gametes

o Triploid 3 haploid sets

Mitosis : no problems

Meiosis : uneven --> infertile gametes

But useful sometimes eg triploid bananas with small

infertile, edible seeds

Mechanism

Failure of chromosome separation during mitosis

Autopolyploidy
o Polyploidy from single ancestor set of chromosomes
o Autotriploids can be produced experimentally by crossing
diploids with tetraploids (gametes n + 2n = 3n); used
commercially

in

some

bananas,

potatoes,

seedless

watermelons
o Tetraploidy

produced

experimentally

using

colchicine

which inhibits microtubule formation or cold/heat shock

(Fig 8.9)

Allopolyploidy
o Chromosome sets from 2 or more ancestral species
(hybrids from distant diploid species)
o Hybridization

&

allopolyploidy

important

in

plant

evolution & breeding

Endopolyploidy
o When certain cells in an otherwise diploid organism are
polyploidy eg. human liver cells

4.2.2 ANEUPLOIDY

42

Polysomy - extra chromosome copies, not sets abnormal

phenotypes
o Trisomy 2n +1

Monosomy - missing chromosome copy harmful, often lethal

2n-1

4.3 NORMAL KARYOTYPES

Chromosome type & composition = karyotype

Human Male = 44 autosomes + XY

Human Female = 44 autosomes + XX

Usually stained by Giemsa = G Banding

Arranged in pairs 1 --> XY

The

analysis

of

stained

chromosomes

with

respect

to

chromosome number and structure is a discipline referred to as

cytogenetics

4.4 ABNORMAL CHROMOSOMES

4.4.1 ABNORMAL KARYOTYPES

Trisomy 21 - Down Syndrome

o Non-disjunction 21, usually oogenesis, associated with
age of mother
o 1/800 live births
o Also Robertsonian translocation in familial cases

Features heart malformations, malformations of the GIT,

mental retardation, characteristic facial features, increased

incidence of leukemia (10 20x higher), hearing loss, many develop
neuropathology of Alzheimer disease

43

Mothers age and risk of Down Syndrome in child

20-29 ~ 1/2000
30-39 ~ 1/600
40-44 ~ 1/100
45-49 ~ 1/40
fetal loss rate = ~30% of Downs syndrome

Trisomy 18 - Edwards Syndrome

o 1/8000 live births
o Survival average 3 months, multiple defects, 1% reach
age 10.
o 48% fetal loss rate

Trisomy 13 - Patau Syndrome

o 1 in 19000 live births
o Survival average 3 months, cyclopia, proboscis,
multiple defects
o 69% fetal loss rate

Duplication

larger

chromosomes

usually

not

viable

miscarriage

Non-disjunction sex chromosomes XO (Turner)

XXY (Klinefelters),

XYY (XYY male),

XXX (Triple X female)

4.4.2 CHROMOSOME ABERRATIONS

Deletions
o Harmful
o Larger deletions most harmful

44

Duplications
o Less harmful than deletions
o But larger duplications --> less survival

Inversions
o Reversed order of genes can unequal crossing-over

Translocations
o Exchange of chromosome parts in non-homologous
chromosomes = chromosome rearrangement

Fragile sites eg. Fragile X

o Mental retardation, variable
o Due to abnormal trinucleotide repeat number which
inactivates nearby gene
o Shows genetic anticipation

Cri-du-Chat Syndrome
An example of a disease caused by chromosomal abberations;

Cat Cry
o

voice abnormalities, multiple oddities

retardation and muscular defects

Due to deletion of small part chr 5 ie. 46, 5p-

4.4.3 CHROMOSOME ABNORMALITIES ASSOCIATED

Abnormalities associated with cancer types

Eg. Philadelphia chromosome

WITH

CANCER

o C22 + C9 + (9;22)
o Part of 22q translocated to 9
o Associated with particular type of leukemia (CML)

Often breakpoints at oncogenes

45

Eg. abl oncogene + (9;22): leukemia

Eg. ras oncogene 11p-: Wilm's tumour

Eg. myb oncogene + (6;14): lymphoma

Eg. mos oncogene + (8;21): leukemia

Eg. myc oncogene + (8;14): leukemia

4.4.4 HUMAN GENETICS

Frequency Of Birth Defects

Dominant & X-Linked
Recessive

1.0
0.1

Chromosomal

0.4

Congenital Malformation

9.0
10.5%

Therefore 10.5% human newborns carry genes --> disorders

during lifetime

9% - malformations such as cleft lip, palate & polygenic

disorders eg diabetes, ulcer etc developmental disorders

1.5% - Mendelian disorders & chromosomal abnormalities

Infants and Infant Deaths (Parents would be candidates for

a genetic referral):

3-5% of all births result in congenital malformations

0.5% of all newborns have a chromosomal abnormality

7% of all stillborns have a chromosomal abnormality

20-30% of all infant deaths are due to genetic disorders

30-50% of post-neonatal deaths are due to congenital

46

malformations

Single Gene Disorders

1986 - McKusick, V: Mendelian Inheritance In Man; contained

4000 Mendelian traits, most associated with disease

Autosomal Dominant

: 50%

Autosomal Recessive

: 43%

X-Linked

: 7%

EXAMPLES
Cystic Fibrosis
PKU

AR
AR

Glu-6P-DHase
Familial

1/10000
XL

AD

1/2500
1/1000

1/500

Hypercholesteremia
Thalassaemia

AR

1/16000

Haemophilia

XL

1/8000

Often frequency hard to determine due to:

o Penetrance
o Variable expressivity
o Heterogeneity many syndromes with similar symptoms;
different mutations same disorder
o Also, treatment & early intervention can affect frequency, eg.
PKU: early intervention (diet) allows individuals who are
affected to live & reproduce
o Others have frequency changes due to pressure (selection)
against reproduction or reduced fertility

47

Multigene Disorders

Some genetic disorders due to several genes interacting =

polygenic

Some complex - polygenic plus environmental factors

o Eg. Hypertension, diabetes

Some disorders show familial aggregation, indicating a genetic

involvement
o Eg. Spina bifida - risk increases with previous child affected
child or parent (to 1/20) also for anencephaly, with a previous
affected child increases risk to 1/20

Spontaneous Miscarriages

1st Trimester

50% chromosomal abnormalities

XO (Turner), 20% of miscarriages total (95% Turners

miscarried)

Autosomal trisomies = 50% abnormal chromosome conceptions

o Many never seen liveborn

Prevalence Of Genetic Conditions

20-30% of all infant deaths are due to genetic disorders

30-50% of post-neonatal deaths are due to congenital

malformations

0.5% of all newborns have a chromosomal abnormality

7% of all stillborns have a chromosomal abnormality

11.1% of paediatric hospital admissions are for children with

48

genetic disorders

50% of individuals found to have mental retardation have a

genetic basis for their disability

12% of adult hospital admissions are for genetic causes

10% of the chronic diseases (heart, diabetes arthritis) which

occur in the adult populations have a significant genetic
component

Robinson A. and Linden MG. 1993. Clinical Genetic Handbook,

Boston, Blackwell Scientific Publications.
Weatherall DJ. 1985. The New Genetics and Clinical Practice,
Second Edition. Oxford: Oxford University Press.

4.5 GENETIC DIAGNOSIS

Symptoms, family history

Determination of mode of inheritance

Determination of risk to preclinical, prenatal situations

Therefore Risk Assessment

Genetic counselling involved

4.5.1 PREIMPLANTATION TESTING

For IVF

In recurrent miscarriages, maternal age, prior chromosomally

abnormal child or fetus, heritable medical condition, gender
selection

4.5.2 PRENATAL TESTING

49

ULTRASOUND
Can detect developmental defects eg.
o Head -

anencephaly

o Spine -

spina bifida

o Abdomen - polycystic kidneys

o Heart -

congenital heart disease

o Limb -

deformities, shortening

Prevention of disorders by
o Intrauterine treatment in some cases
o Eg. Surgical treatment of urinary tract obstruction or
chemical treatment such as vitamin therapy
o Or termination
SCREENING FOR CHROMOSOMAL ABNORMALITIES

1)

NUCHAL

TRANSLUCENY TEST

~ 12 weeks

Measures thickness of fluid layer at level of foetal neck by

ultrasound

Increase risk of chromosomal abnormalities or heart defects

with increased thickness

+ blood test for maternal free HCG and PAPP-A (pregnancyassociated plasma protein-A) or other markers in second
trimester

HCG higher and PAPP-A lower in trisomies

Risk calculated for chromosomal abnormality in foetus, not

50

diagnostic

If greater than 1/300 and/or patient older than 35YO, diagnostic

tests recommended -chorionic villus sampling or amniocentesis

2) CHORIONIC VILLUS BIOPSY

~12 wks

Sample taken trans-abdomen under US guidance

But 1/100 risk of miscarriage

Also chance of sampling maternal cells and not detecting

abnormality

Cells separated & used for testing, FISH for trisomies and X Y,
karyotyping, biochemical testing

3) AMNIOCENTESIS

14-17 wks

Sample of amniotic fluid containing foetal cells through

abdomen

Foetal cells must be cultured for cytochemical tests or used for

biochemical or DNA testing

Results take longer

1/200 risk of miscarriage

More accurate as only foetal cells sampled

3) CELL FREE FOETAL DNA

Non-invasive

From plasma of pregnant women

Not as accurate

51

TESTS PERFORMED

ON

FOETAL CELLS

A)

FISH

Cytochemical testing

Fluorescently labelled probe hybridises to fetal chromosomes

Can pick up trisomies, Turners monosomy

B) KARYOTYPE ANALYSIS

Cytochemical testing

Culture fetal cells, arrest in metaphase on slides, stain, observe

microscopically for abnormalities

C) BIOCHEMICAL TESTING

Better to use amniocentesis & analyse fluid for:

Inborn errors of metabolism eg PKU

Structural protein disorders eg osteogenesis imperfecta (fragile

bone disorder)

Neural tube defects eg spina bifida by chemical monitoring

D) DNA TESTING

By direct mutation analysis of genes, eg. Thalassaemia

By analysis of linkage markers eg Huntington's

Therefore defect known or linked (defect close to marker)

Detect DNA sequence changes by Southern Blot or PCR

RFLPS

52

Restriction Fragment Length Polymorphisms

DNA sequence changes in restriction enzyme sites

Stable, Mendelian inheritance

Can occur within genes or within non-coding regions

Useful DNA markers

Alleles can be tested for linkage or association

Detect by Southern Blotting

Or PCR (useful for RFLPs involving insertions or deletions)

RFLP may cause defect such that individuals inheriting one

allele will get disease, eg sickle-cell anaemia

Or RFLP linked to disease ie DNA containing RFLP on same

chromosome eg X-linked muscular dystrophy

3.5.3 NEONATAL TESTING

Guthrie Test

Heel prick blood test

Test for PKU (Bacterial inhibition assay), CF (PCR and mutation

analysis) and other
disorders

Early detection useful for prevention of eg mental retardation

and genetic counselling

3.6 TREATMENT
1. Dietary Exclusion
Eg. Phenylalanine in PKU
2. Dietary Addition
Eg.

Uridine

supplement

in

orotic-aciduria

(abnormal

pyrimidine metabolism)

53

3. Drug Avoidance
Eg. Glu-6P-DHase - antimalarial drugs & fava bean cause RBC
haemolysis
4. Body Elimination
Eg. Haemochromatosis, iron accumulation --> heart, liver,
pancreas disorders
Remove iron by venesection
5. Cofactor Supplementation
Eg. Enzyme error, increase cofactor eg B12, B6 cause enzyme
to act normally
6. Enzyme Repression or Inhibition
Eg. Supply substrate stopping overproduction of metabolite by
feedback inhibition
7. Replacement Therapy
Eg. Blood in Thalassaemia
Factor VIII in haemophilia, missing enzymes, hormones
8. Organ Transplantation
Eg. Cystic disease, kidney transplantation
9. Preventative Surgery
Eg. Removal of colon in hereditary colon cancer (during early
stages)
10. Preventative Therapy
Eg. Anti-Rh antibody injection into Rh- mother after birth of 1st
Rh+ child
Kills Rh+ blood cells before immune system is stimulated
(injection decays before next pregnancy

54

55

Topic 4 DNA Technology

3.1 RECOMBINANT DNA TECHNOLOGY
Recombinant DNA refers to the joining of DNA molecules,
usually from different biological sources, that are not found
together in nature
Recombinant DNA technology is used to isolate, replicate, and
analyze genes
The introduction of recombinant DNA technology has allowed
genetic engineering
Basis is technology of making recombinant molecules

Inserting DNA into vector & making multiple copies, ie.

Cloning

3.1.1 CLONING STEPS

1. Cut donor DNA
2. Cut vector DNA
3. Join fragments
4. Introduce into host
5. Screen for desired gene

3.2 TOOLS

3.2.1 Restriction Enzymes

Most useful enzyme for DNA work are Restriction Enzymes (RE)

also called restriction endonucleases

These are enzymes that cut DNA at specific nucleotide

56

sequences
Act as means of removing foreign DNA in bacterial cells

Bacteria have a modification system that methylates own

chromosomal DNA to stop RE digestion

3.2.2 Recognition sites

Most

recognition

sequences

exhibit

form

of

symmetry

described as a palindrome, and restriction enzymes cut the DNA

in a characteristic cleavage pattern.
palindromes, eg |
GAATTC
CTTAAG
|
Mostly four to six nucleotides long
Some contain eight or more nucleotides
Cut to give blunt or sticky ends

3.2.3 OTHER ENZYMES

1. DNA Ligase

Joins DNA molecules

2. Alkaline Phosphatase

Removes 5`-P, stops re-ligation

Useful after RE digestion to stop self-ligation

3. DNA Polymerase

Copies SS DNA to give DS DNA

4. Reverse Transcriptase

Makes DNA from RNA template

57

Useful for cDNA library construction

5. Terminal Transferase

Useful for 'tailing' DNA fragments ie attaching bases at

3`end to create sticky ends

3.3 VECTORS
Vectors are carrier DNA molecules that can replicate cloned
DNA fragments in a host cell
Wide variety generally derived from plasmids, phage
Many new artificial constructs

3.3.1 DESIRED CHARACTERISTICS

Capable of autonomous replication in host cell
Small size (usually 3-7kb) generally
Normally capable of amplifying cloned sequence
At least one unique RE site for insertion
Markers

for

easy

identification

&

selection

(Antibiotic

resistance, colour, fluorescence)

Appropriate signals of expression desired (Colour, fluorescence,
linked gene metabolism)
Vectors: Many Types Include:
1. Plasmids
2. Lambda Phage
3. Expression Vectors
4. Cosmids
5. Shuttle
6. YAC
Choice of vector depends on:

58

Type of experiment

Size of insert

Available RE sites

3.3.2 Plasmids
Small, circular, self-replicating
Extra chromosomal
1-many copies/cell (amplification)
Other genes for drug resistance = genetic markers
Most widely used are pBR322 & derivatives

Many single RE
sites; tet and
amp markers

An artificial construct

Inserts: up to 15kb (5-10kb stable inserts)

Select recombinants by insertion into antibiotic resistance

gene

Eg. Cutting with PvuI or PstI, then will be tetracycline

resistant but ampicillin sensitive

3.3.3 BACTERIOPHAGE LAMBDA ( )

E.coli virus
49 kb circular structure

59

 Single RE insertion sites or double sites for replacement DNA

Most commonly used types are

gt10

- all purpose vector

- if form plaques, recombinant

gt11

- expression system

- if insert --> hybrid gene, select by antibiotics

Inserts : 10-20 kb (<23kb)
Lambda contains essential genes + a replacement region. Linear
molecule circularised by annealed cohesive ends - cos site
Select by plaque formation - if no replacement region or DNA
insert here with plaque formation

3.3.4 BACs
Bacterial Artificial Chromosomes
Low copy number plasmids
Good for large inserts
Can accept 100-300kb of DNA

3.3.5 YACs
Yeast Artificial Chromosomes
Replicate & segregate STABLY during mitosis & meiosis
Smaller than 50kb unstable, therefore insert >50kb
2 origins of replication

ori - bacterial origin

ars - autonomous replicating sequence (yeast)

Other structural features

CEN - centromere sequences, attachment sites for spindle

TEL - telomere sequences for integrity of chromosome ends

Selectable markers

60

ampr (plasmid)

TRP1 URA3 [grow in TRP- (tryptophan) URA- (uracil) yeast

host cell]

3.3.6 Expression Vectors

These contain promoters that allow expression of gene products
These contain origins of replication, for replication in E. coli.
Usually contain selectable markers to select transformants in
bacteria (E.coli) (eg AmpR) and eukaryotic cells (G418/Neomycin).
Contain a multiple cloning site (MCS) downstream of the promoter
to insert DNA to be expressed.

3.4 HOST CELLS

FOR

CLONING

Bacterial cells
Yeast cells; grow like bacterial cells but eukaryote; can modify
protein; genetics well known; safe for production of vaccines etc.
Plants
Insects
Human cell lines

3.5 RECOMBINANT DNA LIBRARIES

Collection of cloned DNA sequences from a single source eg.
Cell type, tissue type, individual

3.5.1 Library Construction

1. Cut source DNA
2. Select & cut vector
61

3. Ligate desired DNA into vector

4. Select recombinants
5. Screen for desired clone eg. probes

3.5.2

GENOMIC

VS CDNA

LIBRARIES

Genomic - all DNA sequences from source, coding & non-coding

cDNA - only sequences used in making mRNA transcripts,
therefore only coding DNA from source
For genomic libraries, tend to use partial digests to get
overlapping fragments to get intact gene in at least some
fragments
Also allows 'walking' to get to desired gene
To represent library fully better to insert into vector that can
take bigger DNA fragments, that way less clones to screen for
desired gene Eg. phage or cosmid rather than plasmid

3.5.3

CDNA

LIBRARY CONSTRUCTION

Isolate mRNA

62

3.5.4

LIBRARY SCREENING

Identify recombinant containing desired insert by hybridization

to labelled SS probe
63

 Probe may be partial sequence oligonucleotide or related

sequence from similar species or related gene (eg pseudogene)

3.6 MOLECULAR GENETIC TECHNIQUES

3.6.1

PCR

Polymerase Chain Reaction

Process for producing large amounts of a specific DNA
fragments in vitro, enabling use of this DNA sequence for variety
of experiments
Developed in 1983 by K.Mullins & F.Faloona - started use 198586
Requires
Template - DS DNA or RNA
2 oligoprimers (not complementary)
dNTPs, buffer, Mg2+ Taq Polymerase
NEED:

Sequence known at ends of region to be amplified

Sequence for amplification

PCR Method
------------------------------------------- DS DNA
denature, anneal oligoprimers
----------------------|||
<~~~
~~~>
|||
----------------------Taq Polymerase
----------------------|||
~~~~~~~<~~~
64

Cycle 1

2 DS DNA
~~~>~~~~~~~
|||
----------------------denature, anneal, Taq...
----------------------~~~~~~<~~~~
~~~~>~~~~~~
----------------------4 DS DNA
----------------------~~~~~~<~~~~
~~~>~~~~~~~
----------------------... and so on (up to 30 cycles ~ 230)

65

PCR Points
TAQ POLYMERASE
DNA Polymerase isolated from Thermus aquacticus
Not denatured by heating to 90-95C
Optimum temperature 75C
AMPLIFICATION PLATEAU
30-35 cycles
= 230-235 copies DNA sequence
No further amplification
Cycle - 3 steps
(i) Denaturing

DS DNA ---> SS DNA

95C, 30 secs

(ii) Annealing

Anneal primers to SS DNA

1 minute 37-65C

(iii) Extension

SS --> DS DNA

1 min; 60-72C

Taq Polymerase - allowed automation

Enzyme can be cooled repeatedly heated & cooled

High temperature optimum --> high stringency

Primers - not complementary

If were could hybridise to each other

PCR can be used to amplify genes or non-coding fragments

Eg. Detect insertion/deletion/mutation by PCR

Extract DNA, PCR

Run DNA on gel

Observe bands

Larger or smaller bands depending on size of DNA

66

between primers

3.6.1

REAL-TIME PCR

Allows quantification of gene expression

Several different types eg. RT-PCR or quantitative real-time PCR
In quantitative real-time PCR mRNA is converted to cDNA
Fluorescent dyes and probes are used which bind to the DNA
between the primers; extension causes emission of light which is
detected by a laser
Greater fluorescence = higher gene expression

3.6.2

ELECTROPHORESIS

Gel electrophoresis

OF

NUCLEIC ACIDS

is transfer through a porous

gel or

membrane under charge.

Submarine gel electrophoresis is conducted in a submerged

gel (under buffer).

Capillary gel electrophoresis is conducted in a capillary

Separation of molecules based on size & charge (eg protein gel

electrophoresis can also be performed)
Smallest fragments move farthest in the gel
Fragments can be visualized when stained with ethidium
bromide and illuminated by UV light
Standard markers are used to determine size of unknown
product.

3.6.3

SOUTHERN

BLOTTING

Named after Edward M. Southern

Used to identify which clones in a library contain a given DNA
sequence and to characterize the size of the fragments from

67

restriction digest
Transfer of DNA from gel to membrane (usually nylon and
usually positively charged)
Transfer occurs via i) electro-blotting; ii) vacuum and; iii)
capillary
RNA and protein can also be transferred from gel to membrane:
referred to as Northern and Western Blotting respectively.

3.6.4

MICROARRAYS

Function using the same systems as Southern Blot (Probe

annealing)
Chips or slides covered in fixed DNA/RNA probes
Allows detection of concentration as well as presence of specific
sequences.
Recent advances in technology allow multiple millions of probes
on a single chip.

3.6.5

FISH

Fluorescent in situ hybridization (FISH) involves hybridizing a

probe directly to a chromosome or RNA without blotting
FISH can be carried out with isolated chromosomes on a slide or
in situ in tissue sections or entire organisms

3.6.6

DNA

SEQUENCING

DNA - obtain by cloning or PCR

NB: PCR more prone to error (4X more), therefore do not use

68

PCR derived clone. Use direct PCR product to give mixture of

errors
SANGER-(DIDEOXY) SEQUENCING
SS DNA Pol I template
DNA Pol I to extend P32 primer
dNTPs plus ddNTPs

DideoxyNTPs do not form phosphodiester bonds, therefore

stop chain

----> fragments of different lengths 4 reactions, 4 different

ddNTPs
----> gel (polyacrylamide) + autoradiography
MOST COMMON
Animation

at

http://www.dnalc.org/resources/animations/sangerseq.html
NEXT GENERATION SEQUENCING
Often referred to as 454 Sequencing, or Ionic Sequencing
Relies on binding of PCR product/DNA fragment to a bead which
can be isolated in a specific chamber.
Bound DNA to be sequenced is rendered single stranded prior to
sequencing
Sample is then sequenced by sequentially adding one base and a
DNA polymerase and detecting addition (or not) of specific
bases.
Detection can be indirect, by detection of liberated ATP as in
pyrosequencing, or direct, as in detection of H+ released by
base addition, as in ionic sequencing.
Offers advantages in terms of throughput and ability to
determine complex genotypes.
Not currently common, but has potential to overtake Sanger
Sequencing.

69

TOPIC 5 DNA MUTATION

5.1 DNA MUTATION
(Refer text chapter 15)

A mutation is an alteration in a DNA sequence.

Induced mutations result from the influence of an unrelated

factor or agent, either natural or artificial.

Spontaneous mutations happen naturally and randomly, arising

70

from replication errors and base modifications

Mutations can occur in any cell: various changes in phenotype

ranging from minor to major changes

5.1.2 SPONTANEOUS MUTATIONS

Mutations

occur

randomly

but

with

characteristic

rate

depending on:
o Gene
o Organism

Mutations are not adaptive

Eg. Grow bacteria that are sensitive to antibiotic in antibiotic

resistant bacteria
o BUT replica plating showed that mutations already in
these

cells,

ie.

mutations

random:

Lederberg

experiments

Luria-Delbrck fluctuation test also showed that mutations are

spontaneous

Other examples: Insecticide resistance, antibiotic resistant

bacteria, fungus resistant plants

5.1.3 CLASSIFYING MUTATIONS

Various ways to classify

By location
i) Germinal gametes, inherited
ii) Somatic other cells, not generally inherited
iii) Autosomal within genes on autosomes
iv) X-linked within genes on X chromosome

By molecular change
o Frameshift mutation
o Point mutation: nucleotide substitution

71

By phenotypic effects
o Lethal mutations
o Conditional mutations - phenotype changes, dependent on
environment

eg temperature sensitive Siamese cats fur colouring

5.1.3.1 POINT MUTATIONS

Aka. Base substitutions Eg. An A substituted by a C

MAY be non-coding or coding, which may result in change in

codon which may change an amino acid (missense mutation) or
may not (silent mutation), OR lead to chain termination
(nonsense mutation), if altered to UAA, UAG or UGA

Usually no physiological effect, but occasionally there may be

Eg 1. Haemoglobin Variants
DNA
mRNA
Protein

GGA CTC CTC

CCU GAG GAG
Pro Glu Glu

DNA
Hb mRNA
cell anaemia
Protein

GGA CAC CTC

CCU GUG GAG

Hb

Pro

Val

- normal

has

sickle-

Glu

Eg 2. FOP Fibrodysplasia Ossificans Progressiva

Two types of point mutations

(i) Transition

Pyrimidine pyrimidine
TC CT

72

Purine purine
GA

AG

(II) TRANSVERSION

Pyrimidine purine or vice versa

o 8 variations

Spontaneous mutations of this sort biased to transition because

of 'wobble' ie 3rd nucleotide of codon can vary giving same
amino acid & often transition mutation gives same amino acid =
Silent Substitution

5.1.3.2 Frame-Shift Mutation

Insertion or deletion of a nucleotide can major aa change

Through alteration of the reading frame

o Eg. Deletion

she ca(n) scr eam lou dly

she cas cre aml oud ly.

o Also possible, new aa is STOP codon short protein

Eg. Duchenne muscular dystrophy, ABO blood system

5.1.3.3 MUTATION TYPES

1. Silent - no change to aa sequence
2.

Missense

substitution

changed

aa

sequence

eg.

Achondroplasia
3. Nonsense - substitution Stop codon eg. Marfan syndrome

73

4. Addition/Deletion - add or remove one or more bases

Often occurs with short tandem repeats or runs of particular
nucleotides eg. Cystic fibrosis
5. Frameshift - addition/deletion can change reading frame
6. Transposon Mutations - inserts into gene
7. Expansion of repeat sequences eg. Fragile X, Huntington Disease

Some diseases can arise from a large number of mutations

Eg. Beta thalassemia

5.1.3.4 BOMBAY PHENOTYPE

Affects inheritance of the ABO blood type

A rare recessive mutation in the FUT1 (fucosyl transferase)

gene, involved in synthesizing the H substance, prevents the
addition of the terminal sugars of the A and B alleles

Individuals are functionally and phenotypically Type O although

genotype may be A/B

5.2 PROCESSES

DNA replication errors

Base mispairing

Tautomeric shifts in nucleotides

Depurination, deamination

Oxidative damage

Integration of transposons

5.2.1 TRANSPOSABLE ELEMENTS

Found in prokaryotes and eukaryotes

Jumping genes
74

Can create mutations or silence genes by insertion into

functional genes or alter expression

Can trigger duplications, deletions or translocations

May be germ-line

Involved in human disease

5.3 INDUCED MUTATIONS

1) Base Analogs

Compounds similar to bases

Cause mutations because prone to mispairing

o Eg. 5-bromouracil - T analog but can change to enol form
& pair with G (instead of A) during replication

Next Replication GC formation

2) Alkylating Agents

Add chemical groups to bases different pairing or base

distortion
o Eg.

Nitrosamines,

nitrogen

mustard,

ethylmethane

sulfonate (EMS)
3) Intercalating Agents

Ethidium bromide, Chemotherapeutic agents

Insert between adjacent bps

o Misaligned template/daughter stands replicated DNA
o Addition/deletion in daughter strands

3) Adduct forming agents

mutation-causing

chemicals,

bind

to

DNA

and

alter

its

conformation, thereby interfering with replication and repair

75

Acetaldehyde
o Component of cigarette smoke

Heterocyclic amines (HCAs)

o Cancer-causing chemicals created during cooking of meats
(beef, chicken, and fish)
o 17 different HCAs linked to cancers of stomach, colon, and
breast

4) UV (Ultra Violet)

UV dimer formation

Transcription + replication blocks

5.3.1 RADIATION

X-Rays - dental, medical

Alpha, beta, gamma rays - radioisotopes, fallout,

UV - lower energy, Sun

Ionizing Radiation

DNA Damage

SS break, sugar-P-backbone

DS break

Base alteration
o Last two - most damaging

Also

chromosome

breaks

in

eukaryotes

translocations,

inversions, duplications, deletions

Amount of damage depends on:
i) Total Dose
Mutation rate directly proportional to dose received in Drosophila

76

R = Roentgen = units for exposure dose

ii) Dose-Rate

Same rate dose given quickly is more damaging than given

slowly

Eg. Mice 90R/min - 4X more mutations as 90R/week for same

total dose
o Suggests some repair mechanisms
o But no safe level, ie. threshold dose below which all
damage repaired

DOUBLING DOSE

Humans, 45R X-rays; doubles the mutation rate

o 1960 - chest X-ray : 0.1R
o 1980 - chest X-ray : 0.01R)

TUMOUR THERAPY RATIONALE

Interphase cells less susceptible to chromosome break

Therefore cancer cells more mitosis

Therefore

more

lethal

chromosome

breakage

induced

by

radiation
5.3.2 DNA POLYMORPHISMS

Normal variations in the genome

Defined as occurring in >1% of population

Arise point mutation

RFLPs : RE site

77

5.4 DNA REPAIR

DNA repair systems maintain the integrity of genetic material
susceptible to spontaneous and induced damage, protecting against
disease and cancer
5.4.1 TYPES

OF

DNA REPAIR SYSTEMS

Different enzyme systems available for repair of specific errors. Not

all available in humans.
MISMATCH REPAIR

System for detecting & correcting after proofreading

Uses excision & resynthesis

Mismatched base detected

One strand cut

Region around mismatch excised

o

DNA Polymerase fills gap (uncut strand template)

DNA Ligase seals gap

In prokaryotes, when detect mismatch, enzymes preferentially cut

undermethylated (=daughter) strand to remove error, therefore
sometimes called methyl-directed mismatch repair (methylation
slower than replication)

Error Rates Average

Replication-mismatch: 1 in 10E-5
o ie. Mismatched base for each round of replication
o Chance 1 in 10E-5 wrong

Proofreading - chance of not correcting: 1 in 10E-2

78

o 99% wrong corrected

Mismatch Repair - chance of not correcting: 1 in 10E-3

o 99.9% those remaining corrected

10E-5 * 10E-2 * 10E-3 = 10E-10 (chance that base not correct)

POST-REPLICATION REPAIR

Responds after damaged DNA has escaped repair and failed to

be completely replicated

Recombinational Repair

Break in DS DNA

Rec A protein binds, initiates strand exchange with homologous

DNA double recombination joint

DNA Pol I fills gaps

Recombination joints resolved

Recombination is so that each have one good strand

SOS REPAIR

Last resort

Extreme DNA damage gaps in DS DNA in both daughter

strands

ie. Homologous chromosome damaged

Therefore no recombination repair SOS Repair

~ 20 SOS genes which insert bases into gaps without template

therefore 3 out of 4 bases wrong

Extensive mutagenesis

PHOTOREACTIVATION REPAIR

79

Light dependent

Removes thymine dimers caused by UV light.

The process depends on the activity of the photoreactivation

enzyme called Photolyase

Photolyase captures energy from light and breaks covalent

bonds between thymine dimers

Humans and other organisms lack photoreactivation repair

EXCISION REPAIR

Light independent

Exists in all prokaryotes and eukaryotes

Damaged region cut out by nuclease, gap filled by DNA Pol &
sealed by ligase

Base excision repair removes bases

Nucleotide excision repair (NER) removed bulky lesions

Human auto-recessive diseases that occur due to failure of NER

pathway
1. Xeroderma pigmentosa
o Lost ability to undergo NER
o Affected individuals exhibit severe skin abnormalities, skin
cancers, and a wide range of other symptoms, including
developmental and neurological defects
2. Cockayne syndrome (CS)
o Developmental

and

neurological

defects,

sunlight

sensitivity, but no increase in cancers

o Premature aging and death by age 20
3. Trichothiodystrophy (TTD)
o Dwarfism,

retardation,

brittle

hair

and

skin,

facial

deformities
o Sensitivity to sunlight but no increase in cancers
o Median life span of six years
80

DOUBLE STRANDED BREAK REPAIR

Homologous recombinational repair

Occurs during the late S or early G2 phase of the cell cycle

Exchange of chromosome parts due to breakage & reunion of

intact double helices

Therefore 4 strands involved in cross-over with chiasma (crossover points) visible before Metaphase I

Nonhomologous end joining is activated in G1

5.2 MITOCHONDRIAL DNA MUTATION

For a human disorder to be attributed to mitochondrial DNA,

o the inheritance must exhibit a maternal inheritance pattern
o the disorder must reflect a deficiency in the bioenergetic
function of the organelle
o there must be a specific mutation in a mitochondrial gene

Human mtDNA contains 16,569 base pairs coding for 13 of over

70 proteins required for aerobic cellular respiration

mtDNA is very susceptible to mutations

Doesn't have histones to protect from mutations

Mitochondria have high concentrations of reactive oxygen

species (ROS) generated by cell respiration

ROS damages organelle contents (proteins, lipids, mtDNA)

A zygote receives a large number of organelles through the egg;

a mutation in one or a few will be diluted out by many
mitochondria that lack the mutation and function normally

Heteroplasmy is the condition in which adult cells have a

variable mixture of normal and abnormal organelles

Two disorders arising from mtDNA are

81

o Myoclonic epilepsy and ragged red fiber disease (MERRF) in

which individuals express ataxia, deafness, dementia and
seizures
o Leber's

hereditary

optic

neuropathy

(LHON)

which

is

characterized by blindness

Defective mtDNA is implicated in the aging process

5.5 USE

OF

MUTATION

5.5.1 MUTATION REVERSAL

Wild mutant = forward mutation
Mutant wild = reversion

Basis of Ames Test to test mutagenicity of compounds

uses

any

of

dozen

(mutated)

strains

of

Salmonella

typhimurium selected for their increased sensitivity to mutagens

Therefore

more

lethal

chromosome

breakage

induced

by

chemical

or

radiation
5.5.2 FORWARD GENETICS

Mutants used to determine gene function

Mutagenise

organism

by

mutagen

such

as

transposon

Isolate mutant

Map loci
o Eg. Use complementation analysis to determine if two
mutations causing similar phenotype are in same gene

82

83

TOPIC 6 POPULATION GENETICS

6.1 What is Population Genetics?
-Population genetics the study of genetic variation in groups of
organisms
-Builds on Mendels findings of inheritance of traits in groups of
pea plants due to genetic elements
-Involves study of genetic variation within and between groups to
explain evolutionary forces eg. mutation, selection, migration,
mixing
-Population genetics can help understand human disease patterns
genetic epidemiology
6.2 Human Genetic Variation (the spice of life)
-Genetic variation is important to survival of species because it
allows a population to respond to environmental change. Eg.
Antibiotic and vaccine resistance
-Is the basis of Darwins theory of natural selection.
-Nb. Change in genetic variation over time is slow relative to social
change and fast relative to geographic change
6.2.1 Genomic Variation Among Individuals
-Genomics

is

the

study

of

an

organisms

entire

hereditary

information (DNA makeup)

-No 2 humans have exactly the same genomic makeup (except
identical twins).
-On average the sequences of all human genomes are 99.9% the
same
-This leaves only 0.1% of the genome for sequence variation among
humans.
-0.1% of the genome = 3 million sites (loci).
-The variation at these 3 million loci contributes to variation in
human traits (eg. appearance, disease susceptibility).
6.2.2 Relevance of Genomic Variation
84

-Understanding genomic variation in humans is important for

researchers because;
-DNA variants form the direct basis of heritable traits.
-DNA variants can be used as informative markers in:
-Health and medicine
-Mapping genes for complex disease
-Predicting a patients response to medication
-Forensics science
-Individual identification
-Genetic Anthropology
-Human migration and evolution
6.2.3 Types of Genomic Variants
There are two main types:
1. Single base change events
-Mutations
-Single nucleotide polymorphisms (SNPs)
2. Insertion/deletion of segment of DNA
-Variable number tandem repeats (VNTR)
-Minisatellites (repeat units of 10 500 base pairs)
-Microsatellites (repeat units of 2 6 base pairs)
-Copy Number Variants (CNV)
-copies or deletions of large DNA fragments (>1000bp)
6.2.3.1 DNA Sequence Variants (aka Polymorphisms)
-There are different categories of DNA sequence variations:
-Single nucleotide polymorphisms (SNPs)
agcttctatct
agcttctctct
-Short Tandom Repeat polymorphisms (STRs)
agtctctctctctctctctctctctatacg (CT)11
agtctctctctctctctctatacg (CT)8
-Copy Number Variants (CNVs)

85

cattcaaaggagaaaggagaggtctc
cattcaaaggagaggtctc
>Single Nucleotide Polymorphisms (SNiPs)
-Usually defined as having a minor allele frequency > 0.01 (1%) in
a population
-Most common DNA variants by far!
-Over 2 million have been identified to date.
-In the human genome there is on average 1 SNP every 100 - 300
bp
-Useful for disease gene mapping in human populations.

>Types of SNPs
-Anonymous SNPs SNPs that have no known effect on gene
function. These are valuable as markers only.
-Coding SNPs (cSNPs) SNPs present in the gene coding region of
the chromosome.
-These may represent disease causing variants.
Non-synonymous SNPs SNP results in a different amino acid
(most likely to be functional)
Synonymous SNPs SNP does not result in an amino acid change.
-SNPs are preferred for population genetic analysis over other
types of markers because:
-SNPs are most abundant genetic marker (polymorphism) in the
human genome and provide the best genome coverage
-Modern SNP chip technology allows rapid and inexpensive
genotyping
-Most SNPs are biallelic which makes statistical analysis more
tractable
>Short Tandem Repeats (Microsatellites)

86

-Hypervariable = highly polymorphic due to high mutation rate.

-Up to 40 repeat variations for one locus!
-Spaced approx. 1 every Mb (1,000,000 bases) in the genome
>Copy Number Variants (CNVs)
-Gains or losses (insertions or deletions) of large segments of DNA
1000bp 5million bps
-Can include whole genes and therefore can cause pathology and
human disease
-About 2000 CNVs currently known and may be 100s more!
6.4 Genetic Marker Maps
-Useful for mapping position of genes.
-Useful for disease gene mapping.
-Three main types
i) Cytogenetic Maps (Chromosomal position indicated by cell
staining bands)
ii) Genetic Map (Distance in Centimorgans)
ii) Physical Map (Distance in base pairs)
Rule of thumb 1cM ~ 1Mb
6.4.1 Genome Map of CNVs
-Genomic Variation Databases
-Mutation (Disease) Databases
OMIM www.ncbi.nlm.nih.gov/Omim/
HGMD www.hgmd.org
HUGO www.genomic.unimelb.edu.au/mdi
-SNP Databases
dbSNP www.ncbi.nlm.nih.gov/SNP
TSC www.snp.cshl.org
-CNV Database
TCAG http://projects.tcag.ca/variation/
-Microsatellite Databases

87

GDB www.gdb.org
Marshfield maps www.reseach.marshfieldclinic.org

6.5 Genotype and Allele Frequency Analysis

-Understanding human genomic variation relies on genotype and
allele frequency analysis of polymorphisms.
-Allele = different versions of a genetic locus or DNA sequence
variant (A or a)
-Diploid individuals (humans) have 2 possible alleles
homozygotes = same 2 alleles (AA or aa)
heterozygotes = 2 different alleles (Aa)
-Genotype frequencies = proportion of individuals in the population
having a particular genotype
-Allele frequencies = proportion of alleles in the population

6.6 Probability and Genetics

-Analysis of allele and genotype frequencies relies on probability
laws
-Probability is the chance of an event occurring and values range
from 0 1
-Probability of first child being a boy is 0.5
-Prob of 2 boys is 0.5 * 0.5 = 0.25 (events are independent
product law)
-Prob of 2 boys or 2 girls is 0.25 + 0.25 = 0.5 (events mutually
exclusive sum law)

6.6.1 Probability and Alleles

-In terms of allele frequencies probability that siblings will inherit
identical alleles from parents is independent, therefore multiply
probabilities.

88

-Probability of one child inheriting a disease allele is 0.5 or 50%

chance.
-Probability of 3 children inheriting same disease allele is;
(0.5)3 = 0.125 (or 12.5% chance)
-Probability of 4 children inheriting same disease allele is;
(0.5)4 = 0.0625 (or 6.25% chance)

6.6.2 Calculating Genotype and Allele Frequencies

6.7 Allele Frequencies Vary Among Populations

-In population genetics a population is a group of individuals defined
by some characteristic.
-Group thought to share a common set of genes (alleles)
-Allele frequencies vary in space and time because of evolutionary
forces;
-a new mutation arises spontaneously
-selection occurs when one allele is advantaged (or disadvantaged)
in particular ironment
-migration among individuals occurs through separation and mixing
of individuals
-new populations grow out of a small number of founders (genetic
drift)
-Analysis of allele frequencies helps understand these evolutionary
forces and population histories.
6.8 Relationship between allele and genotype frequencies

89

-Theoretical relationship between the proportion of alleles and

frequency of genotypes was described in 1900s by Hardy and
Weinberg.
-Hardy-Weinberg Law is a mathematical equation for predicting
genotype frequencies from allele frequencies in an ideal population.
-infinitely large
-not subject to evolutionary forces
-random mating
6.8.1 What is Hardy-Weinberg Law?
-Based on probability and binomial expansion rules (see chapter 3)
-For a polymorphism with 2 alleles (A and a)
-Frequency of allele A or f(A) = p
-Frequency of allele a or f(a) = q
p+q=1
(p+q)2=1
-Hardy-Weinberg Law = p2 + 2pq + q2
6.8.2 Hardy-Weinberg Equilibrium (HWE)
-Hardy-Weinberg Equilibrium: p2 + 2pq + q2 = 1
-HWE exists when allele frequencies remain constant from
generation to generation but only under ideal circumstances
-no mutation, selection, migration and large random mating
population
-In reality no population meets the HWE assumptions perfectly but
most human populations do not deviate significantly from the model
6.8.3 Use of Hardy-Weinberg Law
Eg. Albinism is a recessive disorder affecting 1/20000 individuals.
What is the frequency of carriers?
-Hardy-Weinberg Equilibrium: p2 + 2pq + q2 = 1
where,
p2 = normal genotype (AA)
2pq = carrier genotype (Aa)
q2 = albino genotype (aa) = 1/20000 = 0.00005
2
-If q = 0.00005 then q = 0.007
-If q = 0.007 then p = 1 0.007 = 0.993
-Therefore carrier frequency = 2(0.993*0.007) = 0.014
-Therefore 1.5% of population are carriers (~1/70 people)
6.9 Haplotypes analysis is also important in population
genetics
-A haplotype is a sequence of SNP alleles stretching along an
extended segment of DNA essentially a super allele!

90

-Haplotypes are usually inherited as a single unit from parents ie.

same as alleles!
Of course recombination can happen. Haplotype distribution
represents the final product of natural selection.
6.9.1 Association Among Adjacent SNPs in a Population
-If SNPs are close together in the genome alleles will tend to be
inherited together as haplotypes more often then for SNPs that are
further apart.
-Why? less chance that recombination will separate the alleles of
SNPs that are close together ie. break up haplotypes!
-Association of SNPs that are physically close together (ie. alleles on
same haplotype) is called linkage disequilibrium (LD)
Some Practice Questions
-Why study the genetics of populations? give some reasons
-Why is genomic variation important in nature?
-What is the most common type of DNA sequence variant?
-Why do geneticists favour SNPs over STRs? 2 main reasons
-Name and describe a type of genetic marker map
-What is the general purpose of a genetic marker map?
-What is the probability that a couple will have 2 boys and a girl?
-What is a CNV?
-Name 3 evolutionary forces that can cause allele frequencies in a
population to change
-What is the Hardy-Weinberg Law (understand concept, equation
and application)
-What part of the law corresponds to the frequency of heterozygotes
in a population?
-HWE is based on binomial expansion rules (T/F)
-Try doing Problems in the text book
-Define haplotype and linkage disequilibrium

91

TOPIC 7 GENETIC EPIDEMIOLOGY AND

MULTIFACTORIAL TRAITS
Genetic epidemiology
7.1 What is Genetic Epidemiology?
-Epidemiology
-the study of the distribution and determinants of disease in
populations.
-Genetic Epidemiology
-The

epidemiology

of

diseases

with

an

inherited

(genetic)

component.
-Involves surveying the genome!
7.2 DNA Sequence Variations
-There are different categories of DNA sequence variations;
-Single nucleotide polymorphisms (SNiPs)
agcttctatct
agcttctctct
-Simple Tandom Repeat Polymorphisms (STRs)
agtctctctctctctctctctctctatacg (CT)11
agtctctctctctctctctatacg (CT)8
-Insertions/Deletions (indels)
cattcaaaggagaggtctc
cattcaggtctc
7.3 Disease Complexity
-Simple Disorders
-exhibit predictable inheritance patterns Mendelian!
-Usually due to single gene mutations
-rare eg. Cystic fibrosis, sickle cell anemia
-Complex disease
92

-disease

does

not

exhibit

classical

Mendelian

patterns

of

inheritance.
-ie. genotype/phenotype correspondence breaks down
-Due to multiple factors (genetic and environmental)
-ie. multifactorial traits
7.4 Factors Complicating Disease Inheritance in Humans
-Incomplete penetrance individual inherits disease gene but does
not develop disease
-Phenocopy individual has disease but does not have disease gene
(environmental factors)
-Genetic heterogeneity different genes (or alleles) influence same
disease trait
-Genetic Interaction multiple genes join forces to influence the
disease (epistasis)
-Other transmission mechanisms mitochondrial inheritance and
genetic imprinting
7.5 Types of Complex traits
-Continuous Traits eg. measurements such as height, BMI, bp
-follow a distribution with statistical mean and variance parameters
-Discrete Traits
-categorical (meristic) eg. mole count
- threshold eg. Obesity
7.6 How do we know a disease has a genetic component?
-Observation - the disease tends to run in families ie. familial
clustering!
-However, this alone is not enough to conclude that genetic factors
are involved!
7.6.1 Scientific ways of Determining Genetic Involvement in
Disease
-Calculating risk to relatives

93

-Twin/adoption studies
-Calculating Heritability
7.6.1.1 Relative Risk ()
-The magnitude of = degree of familial clustering of disease
indicating genetic component
-Identifying genetic components of disease is much easier for traits
with high values of .
7.6.1.2 Heritability
-How can we quantify the relative contributions of genetics and
environment for a complex disease?
-Use a measurement called heritability (H)
-H estimates the % of the phenotypic variation for a trait that is due
to genes
-Calculating heritability relies on genetic model
7.6.1.3 Phenotypic Variance and the Genetic Model
-Mathematical model describing the relationship among factors
that explain variation in the phenotype ie. variance components
-VPhenotype = VGenotype + VEnvironment + VG*E
-Heritability relates to quantifying V of genetic factors
-2 types of heritability
-Broad Sense (H2)
-Narrow Sense (H)
-Broad Sense Heritability (H2)
-measures contribution of genetic variance (factors) to phenotypic
(trait) variance
H2 = VG/VP
-Narrow Sense Heritability (H)
-more specifically measures contribution of

additive action of

different alleles (additive variance - VA)

94

-VA - thought to be largest main source of genetic variance; others

are dominance variance (VD) and interactive variance (VI)
-VG = VA + VD + VI
-So Narrow Sense heritability, H = VA/VP
7.6.1.4 Twin/Adoption Studies
-Measurements of heritability rely on studies of families, especially
twins
-Can discriminate between genetic or environmental influences on
disease traits
-Adoption studies have shown that the risk of an adoptee having a
disease depends more on biological parents
-Twin studies comparing MZ and DZ twins may show increased
similarity rates of disease (concordance) in MZ over DZ twins
indicating genetic involvement.
Twin Studies (see page 678)
-Monozygotic (MZ) twins derive from single egg and have identical
genotypic make up. ie share 100% of genome
-Dizygotic (DZ) twins have separate eggs and share about 50% of
genome
-The differences in disease concordance values between MZ and DZ
twins can be used to estimate heritability and whether or not a
disease has a strong genetic component.
7.7 Genetic Epidemiology
- The epidemiology of diseases with an inherited (genetic)
component.
-Involves surveying the genome!
7.7.1 Research Approaches in Genetic Epidemiology
1. Candidate gene targeting

95

-Involves targeting specific DNA variants within genes of suspected

physiological importance
-Requires knowledge of disease pathophysiology!
2. Genome Scanning or Mapping
-Utilises adjacent DNA markers in the genome to indicate a region
harboring a disease gene
-Does not require knowledge of disease pathophysiology! A
nave/unsupervised approach!

7.7.2 Study Designs in Genetic Epidemiology

1 Association Studies Tests for associations between DNA
markers and disease traits in unrelated population groups..
2. Linkage Studies tests for co-inheritance of DNA markers and
disease traits in affected pedigrees
7.7.2.1 Association Studies
-Disease-marker association exists when alleles at the marker locus
occur with different relative frequencies in affected and unaffected
(control) patient groups:
-nb see lecture notes for more detail about study design as well as
examples
What about gene-gene interactions
-Typically genetic association studies have focused on only one
gene (variant) at a time.
-For complex diseases any single gene (variant) is only likely to
confer a relatively small effect on the trait ie. has minor clinical
importance
-The combined effect of multiple genes is more likely to be more
important medically.
-Therefore,

strategies

are

required

that

consider

gene-gene

interactions

96

Genome-Wide Association Scans

-Useful when;
-family (inheritance) information is not available eg. DNA banks,
adult onset disorders (usually more common)
-Focuses on unrelated population groups not pedigrees!
-Useful for finding multiple genes each having a small impact on
disease (low penetrance!)
-Eg.

diseases

include

type

diabetes,

Crohns

Disease,

schizophrenia, breast cancer, migraine

-Most popular approach these days because of SNP genotyping
technology
GWAS are based on DNA markers called Single Nucleotide
Polymorphisms (SNPs)
7.7.2.2 Genetic Association Study Design
-To perform GWAS it is estimated that ~500,000 DNA markers
(SNPs) spanning the genome require genotyping.
-1000 cases and 1000 controls may be needed to find low
penetrance genes in GWAS
1 billion genotypes!
-Cost of Genome-wide Association studies?
7.7.2.3 What do we do when we find the genes?
1. Understand the genetic mechanisms that cause the disease
pathology.
2. Determine the strength of disease gene association ie. How well
does genotype predict disease phenotype?
3. Determine how the disease gene association is modified by nongenetic

(environmental)

factors

(ie.

gene-by-environment

interaction).
Some Practice Questions

97

-Definitions of epidemiology
-Compare and contrast simple vs complex disease
-Know the factors that complicate disease inheritance
-Understand section on heritability
-What are the 2 main approaches to mapping genes and what is the
difference?
-What is the definition of association in genetic epidemiology?
-Understand case-control analysis strategy

including testing for

genotype/phenotype association using chi-squared test.

-What has happened to facilitate the whole genome association
scanning approach?
-What do we do when we find disease genes?

98

TOPIC 8 EPIGENETICS
8.1 DEFINITIONS

Epigenetics is the study of gene expression changes that do not

involve changes to the DNA sequence

Epigenetic traits are stably inherited phenotypes that are

transmitted via mitosis and meiosis

The epigenome is the cell specific epigenetic state of a cell

which can be modified throughout an organisms lifetime

8.1.1 EPIGENETIC PATHWAYS

Three categories of pathways that establish and maintain

epigenetic state
1. Epigenators:

environmental

signals

that

stimulate

an

intracellular pathway
2. Epigenetic

initiators:

produce

the

response

to

the

epiginator signals and define the location - include DNA

binding proteins, non-coding RNAs and protein-protein signal
transduction pathways
3. Maintainers: maintain the response once the modifications
have occurred include DNA methylases, histone acetylases
and deacetylases

99

8.2 EPIGENETIC MECHANISMS

8.2.1 METHYLATION

The reversible modification of DNA by the addition or removal of

methyl groups

In mammals, methylation of DNA takes place after replication

and during differentiation of adult cells

Methylation involves the addition of a methyl group catalysed by

methyltransferase enzymes

This occurs on cytosine bases adjacent to guanine called CpG

dinucleotides, which are clustered in regions called CpG islands

CpG islands are located in and near promoter sequences

adjacent to genes

Unmethylated

CpG

islands

adjacent

to

essential

genes

(housekeeping genes) and cell-specific genes are available for

transcription

Other

genes

with

adjacent

methylated

CpG

islands

are

transcriptionally silenced

The bulk of methylated CpG dinucleotides are found in repetitive

100

DNA sequences located in heterochromatic regions of the

genome including the centromere

Heterochromatic

methylation

also

maintains

chromosome

stability by preventing translocation and other chromosomal

abnormalities
8.2.2 X INACTIVATION

One of the X chromosomes in each somatic cell of mammalian

females is inactivated by converting them into heterochromatin
with altered patterns of methylation

This provides dosage compensation for X chromosome genes

Dr Mary Lyon proposed X-inactivation ie. Lyonization in the

1960s

An essential element of Lyon's hypothesis was the random

nature of the inactivation process

Signal from X inactivation centre (XIC) at Xq13 causes

inactivation

Gene called XIST within XIC coats inactive X

8.2.3 HISTONE

MODIFICAT ION

Chromatin is composed of DNA wound around an octamer of

histone proteins to form nucleosomes

Amino acids in the N-terminal region of the histones can be

covalently

modified

by

acetylation,

methylation,

and

phosphorylation

Modifications occurs at conserved amino acid sequences in the

N-terminal histone tails, which protrude from the nucleosome

Chemical modification of histones alters the structure of

101

chromatin,

making

genes

accessible

or

inaccessible

for

transcription

Acetylation by histone acetyltransferase (HAT) opens up the

chromatin structure, making genes available for transcription

Removal of the acetyl groups by histone deacetylase (HDAC)

closes the configuration, silencing genes by making them
unavailable

The sum of the complex patterns and interactions of histone

modifications that change chromatin organization and gene
expression is called the histone code

8.2.4 SMALL

NON-CODING

RNAS (SIRNAS)

siRNAs also participate in epigenetic regulation of gene

expression

After transcription, siRNAs associate with protein complexes to

form RNA-induced silencing complexes (RISCs)

siRNAs can silence genes by interfering with transcription

initiation

siRNAs complementary to promoter regions bind to a promoter

that blocks the assembly of preinitiation complex by preventing
the binding of transcription factor TFIIB and RNA polymerase

Short RNA molecules can also associate with protein complexes

to form RNA-induced transcriptional silencing (RITS) complexes

RITS

complexes

initiate

formation

of

facultative

heterochromatin that silences genes located within these newly

created heterochromatic regions

This is reversible and can be converted to euchromatin, which is

accessible for transcription.

8.3 EPIGENETICS

AND

IMPRINTING

102

Genomic imprinting in mammals results in the expression of the

alleles of a given gene being dependent on their parental origin
ie. some genes are turned on (active) only on the copy that is
inherited from a person's father while others are active only on
the copy from the persons mother. This parent-specific gene
activation

is

caused

by

phenomenon

called

genomic

imprinting.

DNA methylation is a key mechanism of imprinting

Imprinted genes play major roles in controlling growth during

embryonic and prenatal development

IGF2 gene encoding the insulin-like growth factor-2 is an

example of an imprinted gene

In humans (and other mammals like mice and pigs) the IGF2
allele inherited from the father (paternal) is expressed; the allele
inherited from the mother is not.

If both alleles should begin to be expressed in a cell, that cell

may develop into a cancerous cell.

Most human disorders associated with imprinting have their

origins during foetal growth and development eg.

Prader-Willi syndrome occurs due to loss of function of genes in

a particular region of chromosome 15, which are expressed only
in the paternal allele. Common characteristics include abnormal
growth and body composition (small stature, very low lean body
mass, and early-onset childhood obesity), hypotonia (weak
muscles) at birth, insatiable hunger, extreme obesity, and
intellectual disability.

Angelmans syndrome occurs due to loss of function of genes in

a particular region of chromosome 15, which are expressed only
in the maternal allele. It is characterized by developmental
disabilities, seizures, speech deficits, and motor oddities.

Beckwith-Weidmann syndrome is caused by altered methylation

103

of the regulatory regions of genes involved in abnormal growth

such as IGF2. Common characteristics include enlargement of
some organs and tissues, large prominent eyes, large tongue,
seizures.

External or internal factors that disturb the epigenetic pattern of

imprinting or the expression of imprinted genes can have
serious phenotypic consequences

In vitro fertilization (IVF) in humans can cause problems with

imprinted genes.

Children born after IVF and other ART procedures are at risk of
have very low birth weight and have an increased risk of
Angelmans syndrome and Beckwith-Weidmann syndrome

8.3.1 UNIPARENTAL DISOMY

Uniparental disomy (UPD) occurs when an individual receives

both copies of a chromosome from one parent only

Problems occur when the chromosome involved in the UPD is

imprinted

8.4 EPIGENETICS

AND

CANCER

Hypomethylation is a property of all cancers examined to date

For some complex diseases, there are strong links to some

environmental factors, such as smoking and lung cancer

In the 1980s Feinberg and Vogelstein observed that colon cancer

cells had much lower levels of methylation than normal cells
derived from the same tissue

The epigenetic states of normal cells are greatly altered in

cancer cells, and other epigenetic changes, including selective
hypermethylation and gene silencing, are also present in cancer

104

cells

Cancer is now viewed as a disease that involves both epigenetic

and genetic changes that lead to alterations in gene expression

DNA hypomethylation reverses the inactivation of genes, leading

to unrestricted transcription of many gene sets including
oncogenes

While

hypomethylation

is

hallmark

of

cancer

cells,

hypermethylation at CpG islands and inactivation of certain

genes, including tumour-suppressor genes are also found in
many cancers

BRCA 1 is hypermethylated and inactivated in breast cancer and

ovarian cancer

The combination of mutation and hypermethylation occurs in

familial forms of cancer eg. CDKN2A mutation in bladder cancer

Cancer cells also show disrupted histone modification profiles

Mutations in genes encoding members of the histone-modifying

proteins

histone

acetyltransferase

(HAT)

and

histone

deacetylase (HDAC) are linked to the development of cancer eg.

Rubenstein-Taybi syndrome
8.4.1 STEM CELLS

Since

IN CANCER

methylation

transformation

patterns

process,

it

occur
was

very

proposed

early
that

in

the

initiating

epigenetic changes leading to cancer may occur in stem cells

residing in normal tissue

Three lines of evidence support stem cell involvement in

epigenetic changes

1st: Epigenetic changes can replace mutation in silencing

individual tumour suppressor genes or activating oncogenes.

2nd: Global hypomethylation may cause genome instability and

the large-scale changes characteristic of cancer.

105

3rd: Epigenetic modifications are more effective than mutations

in transforming normal cells into malignant cells

The focus of epigenetic therapy is the reactivation of genes that

have been silenced by methylation or histone modification

The FDA in the USA has approved a drug (decitabine, marketed

as Vidaza) for treatment of acute myeloid leukemia and
myelodysplastic syndrome, a precursor to leukemia. It is a
hypomethylating drug.

8.5 EPIGENETICS

Epigenetic
patterns,

AND

changes,
and

BEHAVIOUR
including

histone

alterations

modification

of

may

be

methylation
important

components of behavioural phenotypes

In mice, two regions of the brain show preferential expression of

parental genes

Parent-of-origin effects have been shown in more than 800

genes, supporting the idea that imprinting in different regions of
the brain may represent a major form of epigenetic regulation

In humans, epigenetic changes have been documented in the

progression

of

neurodegenerative

disorders

and

neuropsychiatric diseases with altered behavioural phenotypes

eg. Alzheimer disease, Parkinson's disease, Huntington Disease,
schizophrenia, and bipolar disorder

A controversial theory related to epigenomics concerns the idea

that epigenetic alterations linked to environmental signals
during development or early in life influence behaviour (and
physical health) later in adult life

Several experiments showed that behavioural changes were

mediated by epigenetic methylation of DNA and modification of

106

histones that alter chromatin configuration leading to altered

levels of gene expression

Environments experienced by pregnant animals or newborns

affected the behaviour and health of the offspring as adults.

8.6 EPIGENETICS

Environmental

AND THE

agents

ENVIRONMENT

including

nutrition,

chemicals,

and

physical factors such as temperature can alter gene expression

by affecting the epigenetic state of the genome

The clearest evidence for the role of environmental factors

comes from studies in experimental animals

8.6.1 DIET

A reduced protein diet fed to rats during pregnancy results in

permanent changes in the expression of several genes in the F1
and F2 offspring eg Agouti

In rodents, unmethylated agouti gene = yellow coat colour,

obese and prone to diabetes and cancer, methylated agouti =
brown coat and low disease risk

Obese yellow mice and normal brown mice are genetically

identical but the yellow mice have an epigenetic "mutation. ie.
epimutation

Rodents studies show that exposure to chemicals such as BPA

can also hypomethylate the agouti gene and cause the yellow
obese phenotype

These finding have applications to epigenetic diseases in

humans

Risk of colorectal cancer is linked directly to folate dietary

deficiency and activity differences in enzymes leading to the
107

synthesis of methyl donors

Women pregnant during the 19441945 Dutch Hunger Winter

famine in the Netherlands had children with increased risk of
obesity, diabetes, and coronary heart disease

Hypomethylation of the imprinted IGF2 gene was seen in

children exposed in utero during the Dutch Hunger Winter
compared with unexposed same-sex siblings. The researchers
also found that IGF2 was hypomethylated in individuals whose
mothers were periconceptually exposed to famine, whereas
other genes including leptin were hypermethylated.

The F2 generation also had abnormal patterns of weight gain

and growth

Paternal diet can also cause an effect. Study on an isolated

Swedish community from 1799 onwards based on annual
harvests showed that food abundance during SGP (slow growth
period prior to puberty) was associated with a shortened
lifespan while scarcity of food was associated with an extended
survival of grandchildren

In animal studies epigenetic regulation has been shown to be

induced by both maternal under- and over-nutrition within genes
that control lipid and carbohydrate metabolism and within genes
involved in the central appetiteenergy balance neural network.
In many cases, the epigenetic status of the same genes is altered
both by maternal under- and over-nutrition, although the
direction of the epigenetic change is different.

Several

projects

are

now

focussing

on

how

epigenetic

mechanisms can impact health and control disease processes.

Eg. The NIH Roadmap Epigenomics Project

108

TOPIC 9 BIOTECHNOLOGY
9.1 BIOTECHNOLOGY

Using living organisms to create products to enhance quality of

life

Relies on genetic engineering ie. recombinant DNA technology,

cloning, genomics techniques

Has potential to address global problems ie. health issues, food

shortage BUT

Raises ethical, social, economic questions

9.1.1 RECOMBINANT DNA TECHNOLOGY

Creates artificial DNA molecules from different sources

May be from different species

Several techniques used to introduce foreign DNA into host

genomes

1. DROSOPHILA

Mobile transposon termed the P element,

P element transposons

109

o Central region coding for transposase with flanking 31bp IR

o Transposase section deleted, DNA fragment of interest
inserted & injected into germ cells, along with intact P
element (making transposase) as helper
2. MAMMALS

Usually use retroviral vectors

Inject vector DNA into fertilized egg

Transfer egg to mother for development

Retroviral vector

RNA code for reverse transcriptase that converts to DS DNA &

inserts into host genome Transgenic animals

BUT disadvantages.

Random insertion

Can inactivation of genes

Need inducible promoter to switch on

Prone to rearrangement & deletions

May infect other cells

Other individuals not in need of therapy

Alternatively, alter embryonic stem cells (cells in blastocyst) in

culture by mutating or introduce vector, microinject, into
blastocyst involved in germ line

3. PLANT TRANSFORMATION

Plasmid - large Ti (200kb)

o Contains a transposable element

T DNA element (30kb)

Flanked by 25bp IRs

Plasmid found in soil bacteria: Agrobacterium tumefaciens

Infects susceptible plants (most common flowering plants

110

160,000 species)

Tumours at entry site (usually a wound)

T DNA - codes for protein, stimulating division of infected cells

tumours

Transposase function in plasmid DNA (not T DNA directly).

Plasmid transposase inserts T DNA into plant genome

Can modify to remove tumour genes and add other genes eg.
herbicide resistance

4. SITE-DIRECTED EUKARYOTIC TRANSFORMATION

Early site-directed systems used mutating chemicals favouring

rough areas of DNA

Were random, but useful when specific results not required

More modern system uses zinc finger nucleases (ZFNs)

o Zinc finger nucleases are themselves recombinant proteins
made up of:

Zinc-finger

domains

(specific

sequence

recognition)

Restriction domain (e.g. FokI)

ZFNs are used in pairs to improve specificity

May be directly inserted into target cell or transiently expressed

via plasmid

When bound to target sequence, restriction domain causes a

double strand break

Cell activates recombination based repair to replace damaged

region with alternative copy

Addition of desired alternative sequence in high concentration

facilitates production of specific alteration.

Can include significant insertion of sequence to make fusion

genes, as well as to delete genes or effect single nucleotide
111

changes in a permanent manner.

Therefore has utility in a broad range of genetic engineering

applications

Overcomes many of the problems associated with viral vectors.

BUT o May also result in non-homologous end-join repair

Gene deletion

Oncogenic (cancer causing) fusion genes

May cause apoptosis

o Needs selection to identify properly modified cells

o Delivery to host cells in vivo difficult
o Currently still quite expensive

9.1.2 GENETIC ENGINEERING: APPLICATIONS

Research
o

Isolate gene

Study gene regulation, development, function

Eg. HPRT deficient mice model for Lesch-Nyhan,

Also mouse models for cystic fibrosis, Duchene

Muscular Dystrophy

Gene knockouts: yeast, drosophila, mice

Knockout

gene

in

mouse

cells,

insert

into

blastocyst with different coat colour, mate to

establish homozygous knockout

Production
o

Of particular proteins

Biochemicals, enzymes, drugs, organic chemicals eg insulin,

growth hormone

Commercial

112

New phenotypes

Eg. Bacteria - designed to clean up oil spills, industrial

waste

Eg. Plants - disease resistance, yield

Herbicide resistance (kills other plants)

Gene Therapy - correcting deficiencies, trials underway

Stem Cells tissue engineering, simple grafts and implants in

use

Disease Diagnosis eg. using DNA probes derived from

recombinant DNA methods

9.2 SPECIFIC EXAMPLES

9.2.1 INSULIN PRODUCTION

IN

BACTERIA

Humulin

Precursor polypeptide is preproinsulin, cleaved to active form A

& B chains

A & B subunits inserted into separate vectors beside lacZ gene

LacZ and subunit transcribed and translated as unit = fusion

protein

Fusion proteins cleaved, subunits join = active insulin

9.2.2 PRODUCTION

OF

PHARMACEUTICALS

IN

EUKARYOTES

Limitations of prokaryote hosts: inability to modify eukaryotic

proteins, also misfolding

Eukaryotes hosts including cultured cells or farm animals

Eg. 1-antitripsin, deficiency in heritable emphysema

o Gene

sheep

gene

promoter

into

cloning

vector,

microinject into sheep zygote

113

o Adult sheep produces functional 1-antitripsin

Hepatitis B subunit vaccine

o Gene encoding Hep B surface protein cloned into yeast
expression vector
o Extracted, purified, used as vaccine

Edible vaccines, not yet approved, technical issues

9.2.3 GENETIC ENGINEERING

IN

PLANTS

Prior to genetic engineering, selective breeding used to enhance

Can now add genes to confer resistance to herbicides and

insects

Eg. glyphosphate inhibits chloroplast enzyme EPSP and kills

plants
o Insert bacterial EPSP gene that is resistant to glyphosphate
via Ti plasmid = glyphosphate resistance

Eg. Bt crops: insect resistance

o Bacillius thuringiensis (Bt) protein kills certain insects
o Insert gene into plants = insect resistance
o Controversial because may kill other insect species eg.
Monarch butterflies

Nutritional

enhancement

of

plants

eg.

golden

rice

with

enhanced beta-carotene levels

Concerns: safe to consume?, gene transfer by cross-breeding

with other plants, loss of natural species, built-in sterility (pos vs
cons)

9.2.4

DISEASE DIAGNOSIS

RFLPs restriction fragment length polymorphisms

114

o Polymorphism or point mutation alters cleaving site of

restriction enzyme

ASOs

allele

specific

oligonucleotides,

probes

detect

base

changes caused by point mutations

o Can use for IVF pre-implantation genetic diagnosis

Other methods eg. sequencing

Amniocentesis and CVS allow prenatal sampling for genetic and

other tests

Microarrays and genome scans can test for many mutations in

genome.

Concerns: genetic discrimination, protection of information,

communication

of

results

and

risks,

PGD

and

desirable

characteristics designer babies?

9.2.5 PHAMACOGENOMICS

Reactions to drugs have a genetic component eg. DNA variations

result in liver enzymes with different capacity to metabolize and
use certain drugs

Reactions to certain cancer treatment due to TPMT enzyme

gene variants

Clinicians can tailor dosage after testing for gene variants

Rational drug design synthesises drugs that affect specific gene

products (not trial and error)

9.2.6 GENE THERAPY

Transfer functional genes to patient via vectors, usually modified

retroviral

First trials SCID 1990, ADA gene into T cells

Some

successful

but

problems

leukaemia,

massive

inflammatory response
115

Many clinical trials but none approved yet

Issues to overcome: inactivation of genes, need inducible

promoter to switch on, prone to rearrangement & deletions, may
infect other cells or other individuals not in need of therapy, how
to deliver, multiple gene diseases, how to control expression

9.2.7 REGENERATIVE MEDICINE

Based on genetic modification, or more recently protein

manipulation of cells to form pluripotent or multipotent stem
cells. Some stem cells can be harvested directly.

Stem cells can be induced to form specific cell and tissue types
using signalling factors

Simple cultures may be sufficient for reimplantation (bone

marrow for blood system regeneration in cancer)

Simple structures have been developed for reimplantation

(bone, skin, cartilage, liver)

Problems have been encountered in developing larger and more

complex tissues. Some of these have been overcome in human
and animal trials

Artificial and recycled tissue scaffolds are being investigated to

help with the process

9.2.8 NOVEL PHARMACOLOGICAL USES

Production of humanised antibodies allows targeting of human

proteins by human immune system.
o Humanised anti-VEGF antibody is used for treatment of
cancer and retinal diseases involving blood vessel
growth.

116

o Allows

suppression

of

cells

displaying

aberrant

phenotypes or absorb excess signalling molecules

o Made by adding human elements to specific antibody
gene derived from animal immune response to human
protein and then made in cell culture.

DNA

origami

methods

being

investigated

for

containing

chemicals for delivery, as well as potential nanotechnological

uses.

Adaptamers (DNA fragments binding to specific substrates)

being investigated for drug targeting, cell binding and sensing
proteins.

9.2.9 INDUSTRIAL USES

In addition to biomedical and food production, biotechnology is

beginning to show direct industrial uses.

Bioremediation of toxic substances (oil spills)

Detection of chemicals or other organisms

Fermentation for alcohol production from non-food biomass

Artificial photosynthesis to produce fuel

Biological batteries and power generation

117

TOPIC 10 ETHICS
10.1 ETHICS

The discipline dealing with the consideration of what constitutes

acceptable moral conduct

Major ethical theories:

o Consequentialism:

moral

action

determined

by

consequences
o Deontology: duty-based system of analysis
o Virtue-based: Aristotle; role of inner character traits
o Feminist: caring, empathy and sensitivity
o Religious: varied, can be simple or complex, typically have
many elements
o Principlism: the principles of autonomy, beneficence, nonmaleficence, justice

10.1.1

BIOETHICS

The study of the ethical, social, legal, philosophical and other

related issues arising in healthcare, the biological sciences and
from biotechnology

10.1.2

WHY

IS

BIOETHICS IMPORTANT?

Biotechnology operates in an environment on which past

experiences and current norms may be insufficient to guide our
moral reasoning. The ever-expanding possible applications of
modern biotechnology and the often uncertain consequences
make it imperative to discuss the implications and issues arising
118

from the science.

HTTP://WWW.BIOETHICS.GOV.AU/

10.1.3

BRIEF HISTORY

OF

BIOETHICS

AND

MEDICAL ETHICS

Prior to the Second World War, few nations had laws or even
guidelines for the ethical conduct of human or animal research.

The Hippocratic Oath was often the closest thing to medical

ethics in place.

Following the Second World War, the identification of atrocities

taking place in Nazi concentration camps which included
medical/biological research led to the development of the
Nuremberg Code, the first major document to attempt to codify
medical research ethics.

In 1964, the Nuremberg Code was replaced by the Helsinki

Declaration, which is the current international standard in
describing medical ethics.

Neither the Code nor the Declaration has force in international

law, but most countries with ethical laws and systems in place
use the Declaration to some degree.

Uptake of the principles laid out in the Code/Declaration was not

universal and notable violations did occur after WW2 (e.g. the
1932-1972 Tuskegee Syphilis Experiment)

10.2 PRINCIPLES

OF

MEDICAL BIOETHICS

Autonomy: Actions should not be subjected to controlling

constraints by others

Beneficence: One ought to prevent and remove evil or harm.

One ought to do and promote good. One ought to weigh and
balance the possible goods against the possible harms of an

119

action

Nonmaleficence: One ought not to inflict evil or harm.

Justice: equal access to the goods in life that every rational

person values

(Based on the theory of principlism)

10.2.1

MEDICAL BIOETHICS

IN

AUSTRALIA

National Health and Medical Research Council Act 1992

specifies that NHMRC will issue advice and guidelines on ethics
and related issues in the fields of health and human and animal
research.

Australian Health Ethics Committee (AHEC)

o advises the NHMRC on ethical issues relating to health &
develops guidelines for the conduct of research involving
humans
o members include experts in philosophy, the ethics of
medical research, public health and social science research,
clinical

medical

practice

and

nursing,

disability,

law,

religion and health consumer issues.

National Statement on Ethical Conduct in Human Research

(2007): four principles
1. Research merit and integrity
2. Justice
3. Beneficence
4. Respect

10.2.2

PROCESS

OF

ETHICAL REVIEW

Ethical review for all biomedical research is provided by Human

Research Ethics Committees (HRECs) and Animal Research
Ethics Committees (ARECs).

120

These committees are overseen by the NHMRC and composed of

individuals with scientific expertise as well as members of the
community.

Most Human and Animal Research Ethics Committees in

Australia use the National Ethics Application Form (NEAF) for
their applications, or a modified version.

Applications require the disclosure of all aspects of the intended

research, including:
o Method of recruitment/What animals used
o How many test subjects
o How informed consent is obtained
o What is the scientific basis for the research
o What tests will be done
o What provisions there are for feedback
o How data will be handled
o Who is providing funding
o Where will the research be done
o Who is doing the research

Once application is made, the HREC or AREC will discuss the

merits of the application and reply to the researchers, accepting,
rejecting or requiring modification of the protocols.

Ethical review is for a maximum of five years (three years

initially, with two years extension available). Projects taking
longer than this will need to apply multiple times.

10.2.3

CLINICAL TRIALS

Specifically research aimed at testing a treatment in human

beings.

Some

jurisdictions

also

include

diagnostic

and

observational studies as clinical studies and may have additional

121

subcategories.
o Phase I Safety testing
o Phase II Base efficacy and protocol testing
o Phase III Wide-scale efficacy testing
o Phase IV Post-approval long term studies

Ethical approval for these is somewhat more involved than

standard human research.

More codified protocols are required, with specific requirements

for patient safety and reporting of adverse events.

In Australia, the Therapeutic Goods Administration (TGA) must

also approve a clinical trial before it can begin.

Most HRECs will also require that any clinical trial is registered
in a clinical trial database, such as the Australia and New
Zealand Clinical Trials Register.

10.3 SPECIFIC ISSUES

10.3.1

OF

ETHICAL INTEREST

ETHICAL IMPLICATIONS

OF

BIOTECHNOLOGY

There are many public concerns over numerous issues in

biomedical research and use of biotechnology.

Many of these concerns are based on public perceptions of what

is being done with these technologies, a not insignificant
number

of

which

are

based

on

faulty

or

incomplete

understandings of the science involved.

Nonetheless, there are a number of significant and legitimate

ethical

issues

in

biomedical

research

and

the

use

of

biotechnology.

Among these concerns are:

o Use of animals in medical research (old and ongoing
issue)
o Production of genetically modified food crops

122

o Genetic discrimination
o Use of genetically modified organisms in industry
o Production of artificial life forms
o Stem cell research
o Human cloning

Many fears and concerns have been advanced concerning these

issues. These include potential dangers to human health from
GM foods, the potential to create superorganisms as well as
moral arguments over the use of embryonic stem cell lines.

As scientists, it is our responsibility to ensure that the public is

sufficiently educated about the specifics so as to allow a
reasonable debate on the issues involved.

10.3.2

STEM CELL RESEARCH

Embryonic stem cells: pluripotent, can differentiate & grow in

culture, immune rejection problems, controversial

Adult stem cells may differentiate but not into all cells, difficult
to grow, avoids rejection problems, can turn cancerous, found in
bone marrow, umbilical cords and other tissues

Research in the use of these is progressing constantly. Some

early therapies are beginning to appear now.

Research on mechanisms in embryonic stem cells have allowed

the alteration of somatic adult cells to resemble embryonic stem
cells. Early versions required viral transduction, but the most
recently developed protocols can be induced with proteins only.

Research

into

organ

regeneration

has

made

significant

progression, based in part on earlier grafting techniques.

10.3.3

HUMAN CLONING

123

Somatic cell nuclear transfer (SCNT) was the technique used to

create the first cloned mammal, 'Dolly' the sheep. The nucleus of
an oocyte is replaced with the nucleus of a somatic cell.

The resulting embryo could, in theory, lead to cloned human

beings = Reproductive cloning

Stem cells could be harvested from the cloned embryo to form a

new embryonic stem cell line. The embryonic stem cells could be
encouraged to develop into human tissue or (possibly in the
future) a complete organ for transplant. = Therapeutic cloning

Suggested alternatives
o ANT: Altered nuclear transfer
o OAR: Ooctye assisted reprogramming

10.3.4

STEM CELL RESEARCH

AND

CLONING

IN

AUSTRALIA

In 2002, the Federal Parliament passed the Prohibition of

Human

Cloning

for

Reproduction

Act

2002

and

the

Research Involving Human Embryos Act 2002.

Amended in The Prohibition of Human Cloning for Reproduction

and the Regulation of Human Embryo Research Amendment Act
2006.

The Act states that only embryos that are left over from ART
(Assisted Reproductive Technology) treatments can be used to
derive human embryonic stem cell lines for research. Embryos
cannot be created purely for the purposes of research.

Human cloning is banned in Australia under the Prohibition of

Human Cloning for Reproduction Act.

It is now possible to apply for a license to conduct human

cloning for making embryonic stem cells. The total ban on
human cloning for reproduction will remain.

124

TOPIC 11 Genetic Analysis of Cancer

11.1 DEFINITION
11.1.1

AND

DEFINITION

CAUSES

OF

OF

CANCER

CANCER

Cancer is defined as the abnormal, uncontrolled proliferation of

cells

Tumours can be classified as benign (localised growth) or

malignant (cancerous)

Cancer cells differ from normal cells in 3 ways

They exhibit uncontrolled proliferation that is not

regulated as in normal cells

They grow extensively and invade surrounding tissue

They are capable of metastasis (resulting in formation of

secondary tumours)

Normal cells are under gene control, cancer cells develop due to
mutations in genes controlling basic cellular function

Cancer cells show higher than normal rates of mutation,

chromosomal abnormalities and genomic instability

Cancer cells are clonal

125

They originate from a common ancestral cell that accumulated

numerous mutations

Cancer stem cell hypothesis

Conventional hypothesis vs Cancer Stem Cell Hypothesis

Stem cells have important characteristics that distinguish

them from other cell types

11.1.2

GENETIC CAUSES

OF

CANCER

Changes in genes that normally control cell growth and

differentiation can result in cancer

Genes that control growth and differentiation fit into 2

broad categories: oncogenes (act in a dominant fashion)
and tumour suppressor genes (act in a recessive fashion)

Changes in as little as 1 or 2 genes may result in cancer,

although usually mutations in 6-12 different genes required

Although a particular gene may not be directly responsible for

allowing uncontrolled growth, it may be involved in signalling
pathways

to

indirectly

affect

growth,

additionally

some

susceptibility genes may not be involved in growth but may be

responsible for detoxification of particular carcinogens

Apoptosis (programmed cell suicide) usually destroys cells with

mutations, but sometimes mutations result in the inactivation of
this process allowing damaged cells to proliferate

Oncogenes

Cancer causing genes

Genes whose products are involved in transforming cells in

culture or inducing cancer in animals

May be caused by virus or altered cellular gene

126

Act in a dominant fashion, therefore a mutation in only 1 allele

required to cause cancer

Proto-oncogenes are normal cellular genes that if mutated

(altered) become oncogenes

Proto-oncogenes code for proteins that respond to signals

from other cells to stimulate growth

When mutated to form oncogenes, growth pathways are

continuously active and may resulting in uncontrolled cell
growth

Cancer may result from:

Point

mutations

of

proto-oncogenes

to

become

oncogenes

Chromosome rearrangements such as translocations

may fuse areas of chromosomes together to form
oncogenes

Amplification of proto-oncogenes may lead to increased

expression (therefore behaving as oncogenes)

Double minute chromosomes are acentric chromosome

fragments that may contribute to the development of
tumours by providing amplified oncogenes

Homogenously staining regions are chromosomally

integrated

forms

of

amplified

DNA

containing

oncogenes

Viral insertion may alter promoter regions, increasing

transcription and leading to altered expression

There are approximately 100 known oncogenes, some common

examples are:

Ha-RAS (cellular membrane protein) associated with lung,

bladder, breast, kidney and colon cancers

MYC (factor involved in activating transcription of growth

127

promoting genes) associated with lung, breast, ovarian and

colon cancers

Cyclins (regulate cell cycle) associated with lung and

esophagus cancers, many others

Oncogenes can act almost anywhere on or within the cell,

including cell surface receptors, nucleus, cytoplasm

Tumour Suppressor Genes (TSGs)

Genes that control abnormal cell proliferation

Mutations result in a loss of activity of these genes,

therefore a loss of growth control pathways resulting in
uncontrolled growth

Mutations can be caused by:

Deletions

Point mutations

Methylation at CpG islands

Knudsons two hit hypothesis: TSGs act in a recessive fashion,

therefore mutations in both alleles are required to cause cancer

Sporadic cancers: two somatic mutations in same cell

Familial cancers: one inherited mutation and one somatic

mutation

Examples

TP53 prevents the cell progressing to the S phase of growth

and is likely to play a role in transcriptional control, it is
associated with over half of all cancers including lung, skin,
bladder, breast and brain cancers

RB1 also prevents cell progressing to S phase of growth by

inactivating cellular transcription factors and is associated
128

with retinoblastomas, sarcomas, breast and bladder cancer

BRCA2 involved in DNA repair, associated with breast,

ovarian, prostate cancers.

11.1.3

CELL CYCLE

AND

CANCER

Cancer Cells Contain Genetic Defects Affecting Cell-Cycle

Regulation

Growth and differentiation of cells are strictly regulated.

In cancer cells, many of the genes that control these functions

are mutated or aberrantly expressed, leading to uncontrolled cell
proliferation.

11.1.4

ENVIRONMENTAL CAUSES

OF

CANCER

Many cancers show no form of inheritance and are likely due to

exposure to carcinogens

Exposure to carcinogens usually causes mutations in the DNA

resulting in cancer formation

Examples of this are common:

Lung cancer has been associated with asbestos, smoking,

radon

Skin cancer has been associated with UV radiation, coal tar

and petroleum products and certain drugs and antibiotics

Liver cancer has been associated with PVC (only in

workers), iron overload, alcohol induced liver damage and
arsenic

Bladder cancer has been associated with aniline dyes,

cigarette smoking and ingestion of analgesics or artificial

129

sweeteners

It

is

sometimes

hard

to

distinguish

between

genetic

&

environmental components of specific cancers and many cancers

are due to a combination between genetic predisposition and
environmental carcinogen exposure (causing mutations in cell
cycle related genes)

Age plays a role in many cancers due to an accumulation of

mutations over the years

11.1.5

CLASSIFICATION

Leukemia: blood

Carcinoma:

from

OF

CANCER

endoderm

(gut,

intestines)

or

ectoderm

(epidermis, nervous system)

Sarcoma: from mesoderm (muscle, blood, connective tissue)

11.2 CARCINOGENESIS
11.2.1

AND

METASTASIS

CARCINOGENESIS (CANCER FORMATION)

Cancer is caused by mutations at the molecular level that allow

for uncontrolled growth and differentiation and is characterised
by three steps:

Initiation: a permanent and inheritable change in the cells

genome

Promotion: proliferation of initiated cells

Progression: further genetic alterations that cause genomic

instability and transformation into a malignant phenotype

130

Initiation

May be inherited or caused by environmental factors

Early initiation may be epigenetic, later fixed by mutation

For the initiation process to be fixed (irreversible), one or two

rounds of cell division with the mutation must occur

Initiated cells appear the same as normal cells

Genetic mutation is passed on indefinitely to cells progeny

Most initiated cells remain dormant throughout a hosts lifetime

Promotion

Promoting agents (carcinogens) stimulate the rate of cell division

and can inhibit apoptosis in initiated cells

Promotion is a reversible process

If the promoting agent is removed, clonal expansion will

cease and regression will occur

Increases

likelihood

that

cell

may

be

further

altered

in

progression phase

Progression

May be caused by random somatic mutations or by exposure to

additional

mutagenic

agents

or

epigenetic

modifications

favouring growth

Cells appear different to normal cells

Increased growth rate, invasiveness and metastatic capability as

well as biochemical and molecular changes occur within the cells

131

11.2.2

METASTASIS (CANCER SPREADING)

Metastasis is defined as a neoplasm arising from another

neoplasm that it is no longer in continuity with

In simple terms, it is the spreading of cancer to other areas of

the body by the circulatory or lymph systems

To

undergo

metastasis,

cells

adjust

genetically

and

epigenetically, resulting in a variety of biological properties

Metastasis is an active process, resembling wound healing

The process may commence earlier than previously believed

Successful metastasis involves 6 steps:

Separation

Invasion and intravasation

Circulation

Arrest

Extravasation and invasion

Growth

Separation

Cell detaches from neighbouring cells

Cell must survive without the usual cell to cell interactions

It is believed that tumour cells develop a mechanism, which

breaks down cellular adhesion

Many separated tumour cells are not viable, those that are have
usually made adaptions such as production of growth factors

132

Invasion and Intravasation

Invasion

is

penetration

through

extracellular

matrix

(eg.

Basement membranes, interstitial stroma, cartilage and bone)

into vessels

Occurs through receptor attachment of tumour cell to

tissue, secretion of hydrolytic enzymes, chemotaxis and
motility factors

Intravasation is repeated invasion, many cells move from tissue

to vessels

Often accompanied by angiogenesis (recruitment of new

blood vessels)

Cells may remain in vessels, proliferate and circulate at another

time or may circulate immediately

Role of the extracellular matrix and its constituents

Circulation

Cells enter circulation (blood or lymphatic vessels)

Hostile environment (immunological and mechanical forces

destroy many tumour cells)

Most rate limiting step (<0.01% circulating tumour cells initiate

colonies

Homotypic or heterotypic aggregation may be used as a

defence mechanism to protect a small number of cells

Arrest

Lodging of tumour cells in the vessels of the target organ

133

Mechanical wedging, entrapment or attachment

May be non-specific or site-specific

Invasion and Extravasation

Degradation and movement into the tissue (same as invasion and

intravasation)
*

Success depends on immune status of cancer cells and

induction of angiogenesis

Growth

Tumour growth occurs with the aid of tumour growth factors

Successful metastasis allows for subsequent metastases with

improved efficiency

This may be the limiting factor for successful metastasis, as other

factors may occur early.

11.2.3

CANCER STEM

CELL

HYPOTHESIS

Hypothesis that cancers may harbour a form of stem cell.

Cancer stem cells behave like transformed normal stem cells,

with some cells remaining relatively quiescent while others
rapidly reproduce. The tissue they regenerate is a tumour,
perhaps retaining vestigial function.

Due to quiescence, cancer stem cells are not affected by common

treatments.

They may explain ability of tumours to self renew and be a

component of tumour immortality.

Still somewhat controversial as to the nature of the stem nature

134

of the cells.

Debate as to whether the cells are transformed stem cells,

transformed progenitor cells, or mutated somatic cells that have
regained stem cell-like functions.

11.3 CANCER STUDIES

11.3.1

FAMILY STUDIES

In majority of cases, there is no general predisposition to cancer,

but there is to specific cancers

Breast Cancer

If have 1st degree relative with disease, 10% chance female will
develop by 85yrs (controls 5% chance by 85yrs)

Environmental influence: child-bearing reduces risk of breast

cancer

Stomach Cancer

1st degree relative affected results in 3% chance (control group

1.5%)

Environmental influence: lower socio-economic group increases

stomach cancer risk

11.3.2

TWIN STUDIES

Can be used to determine genetic vs environmental components

Breast & Uterus Cancer

Concordance higher in ID twins, ie more chances of both

siblings affected if identical than non-identical; therefore
larger genetic component

135

Stomach Cancer

Most identical twins discordent; therefore environmental

factors more important than breast cancer but still must be
genetic component since more concordant twins than
general population

11.3.3

ANIMAL STUDIES

Usually use mouse models

Breed strains susceptible to developing particular cancers

Eg. C3H strain (90% or more) develop breast cancer

Development reduced by dietary restrictions

11.3.4

VIRAL STUDIES

In mice shown that milk transmits a virus to newborn that can

greatly increase cancer in cancer prone strains

Evidence of oncogenic viruses involved with specific cancers

Eg. abl oncogene - chronic myeloid leukaemia

11.3.5

BIOCHEMICAL STUDIES

Recent biochemical link genotype & environment with lung

cancer

AHH: enzyme aryl hydrocarbon hydroxylase

Breaks down specific polycyclic hydrocarbons (from tobacco

smoke) to carcinogenic metabolites

Ability to induce this enzyme is variable & genetically

controlled (multi-factorial)

High inducers, greater risk of lung cancer if smoke

136

11.3.6

IN UTERO EXPOSURE

Certain cancers show strong link to in-utero exposure

Eg. Stilboestrol (given to mothers when may abort) ?

vaginal cancer in female offspring

Eg. Phenytoin ? neuroblastoma

11.3.7

MOLECULAR STUDIES OF CANCER

Many studies now on genes & DNA studies involved in cancer

Can involve family based studies on specific mutations or studies

on molecular characteristics and classification

Studies for molecular biology and classification can lead to

improved treatment

Family

based studies give insights

into particular cancer

syndromes or the activity of specific genes

Eg. Retinoblastoma

Auto dominant (40% of cases)

Found enzyme, esterase D, from 13q14, showed linkage in

autosomal

dominant

families

used

to

identify

retinoblastoma gene, which is a tumour suppressor gene

Absence of gene leads to tumour

Found single mutation inherited as auto dominant, with

sporadic somatic event in developing cells of retina (due to
unequal crossing-over as an example)

If not inherited, get double sporadic development, therefore

less likely & later onset & more chance unilateral

11.4 EXAMPLES

OF

CANCER

WITH

PRIMARY

GENETIC

CAUSES

137

11.4.1

POLYPOSIS

COLI

Autosomal dominant polys of large bone

Normally, polyps in the bowel are not a problem, polyposis coli is

a large number of polyps in the bowel, making the person
susceptible to cancer

High risk of polyps becoming cancerous --> 90% affected die of

bowel cancer

RFLPs linked to gene at 5q 21-22

Mutations (single base and small insertions and deletions) within

the gene result in the disease, also different forms of severity

A similar pattern may be seen in skin cancer (solar keratosis ?

squamous cell carcinoma)

11.4.2

XERODERMA PIGMENTOSUM

Auto recessive

Hypersensitivity to UV light

Inability to correct dimer formation resulting in skin carcinoma

(as early as 4-5 years old)

Also ocular and neurological problems

7 XP repair genes have been identified (XPA to XPG)

Defects in these different repair genes result in different

severities of the disease

11.4.3

NEUROFIBROMATOSIS

Auto dominant

Also called Von Recklingkausens' disease

Tumours of peripheral & central nervous system

138

Severity may differ

Two

forms

NF1

(located

chromosome

17),

NF2

(located

chromosome 22)
11.4.4

RETINOBLASTOMA

Auto dominant (in 40% cases), other cases sporadic

Inherited cases: 1 germline mutation, 1 somatic mutation

Sporadic cases: 2 somatic mutations

Both eyes, highly malignant cancer of retinal cells of the eye

Usual onset before 5 yrs

RB1 gene located 13q14.1-2

11.5 TREATMENT
11.5.1

OF

CURRENT

CANCER

TREATMENT

Prevention

Preventing exposure to carcinogens

Eg. Not smoking to prevent lung cancer

Eg. Avoiding sunlight to prevent skin cancer

Done before cancer originates

Chemotherapy

Use of drugs to treat cancer

Commonly used to treat cancers when metastasis is likely (treats

escaped cells)

139

Radiotherapy

Using ionising radiation to treat cancer

Used at some point during treatment of more than half cancers

Damages DNA of tumour but not of surrounding tissue

Surgery

Removal of the cancerous tissue

Preferred method of treatment for some cancers, such as

colorectal cancer

11.5.2

POTENTIAL TREATMENTS

AND

TARGETS

Immunotherapy

Use natural biological substances to activate the immune system

to treat cancer

Three main categories: immune response modifiers, monoclonal

antibodies and vaccines

Can attack cells that have escaped from the tumour (in process
of

metastasis)

and

is

likely

to

be

more

selective

than

chemotherapy

Eg. Breast cancer vaccine

In mice: shrinkage of solid tumours

Trying to replicate in humans

Angiogenesis Inhibitors

140

Angiogenesis is the formation of new blood cells, these feed the

cancer cells with oxygen and nutrients

Natural and synthetic angiogenesis inhibitors may stop the

growth of cancer

In animal studies angiogenesis inhibitors have stopped tumour

formation

Currently being trialled in breast, prostate, brain, lung, ovary

and other cancers

Resveratrol

Resveratrol (found in red wine) has been shown to inhibit

initiation, promotion and progression stages of cancer

Studies performed on mice and cell lines

Preclinical trials

141

Example questions:
1. What are the three key aspect of cancer?
2. How may the cancer stem cell hypothesis affect treatment
strategies?
3. If a cancer cell has WT p53 is this a good or a bad thing?
4. How is cell cycle checkpoint control influenced?
5. How can we determine what phase of the cell cycle a cell is
in?
6. What role does the ECM play in normal tissue?
7. When the ECM is dysregulated, how can this affect cancer
progression? (Give two examples).
8. How is a persons relative risk of cancer determined?
9. What determines treatment?
10.

Can (and how) cancer treatment be improved?

142

Topic 12 Behavioural Genetics

12.1 BEHAVIOURAL

GENETICS

Behavioural genetics aims to identify genes and polymorphisms

responsible for types of behaviour, as well as determining how
they are inheritied and expressed
Behavioural traits are incredibly complex to unravel due to
multifactorial genetic and environmental factors which affect
behaviour:

Phenotypic variance (VP) is the variation in a trait or

behaviour among individuals in a sample or population.

Genetic variance (VGen) is the proportion of phenotypic

variation due to genetic factors; can be additive (VA) where
alleles have cumulative effects, or non-additive (V D) where
dominance or epistasis can influence the phenotypic
variance.

Environmental

variance

(VEnv)

is

the

proportion

of

phenotypic variation due to environmental factors; can be

common to family members (VC) or non-shared (VE).

Gene-environment interactions can also contribute to

variance (VGXE); this is when the environment only has an
effect on VP in the presence of certain genotypes, and vice
versa.

Gene-environment correlations can also contribute to

variance (rGE); this is when certain environments and
certain genotypes are correlated (tend to appear together).

143

 Multiple study types are required to get complete picture of

genes involved in complex behaviours, including:
1. Animal studies
2. Family/twin/adoption studies
3. Case-control association studies
Each study design can reveal specific information about the
genetic influence on the studied trait

12.2 ANIMAL

STUDIES

To use animals in studies of human behaviour, need to examine

whether the model is valid for the disease being studied:
1. Predictive validity: how well does the animal model predict
the human condition?
2. Construct validity: does the model accurately measure what it
intends to measure?
3. Face validity: does the animal model reproduce symptoms of
a human condition?
The following aspects of the animal model must be examined for
validity: choice of species, choice of model, choice of test.

12.2.1 Choice of species

To select a valid species for an animal model, need to consider
the relation of the model to humans.

Obvious differences in anatomy/function are expected but

animal model must have sufficient common structural
organization and homology to humans in the organs,
tissues, or systems under study.

144

 Two main types of models for behaviour, invertebrate and

vertebrate animal models

1) Invertebrate animal models

Commonly used invertebrates include C. elegans (nematode)

and Drosophila (fruit fly)
Invertebrates have a disadvantage of lesser homology with
humans than vertebrates, but huge advantages of greater
practicality.
They can still be used as a valid animal model if there are
behavioural

responses

which

parallels

those

observed

in

mammals i.e. Drosophila exhibits many complex forms of

behavior

including

courtship,

feeding,

circadian

rhythms,

learning, memory
Drosophila is the most commonly used due to many advantages:

short generation time

small, easy and inexpensive to culture

high fecundity (breeding rate)

males and females are easily distinguished

well-characterised genome

2) Vertebrate / Mammalian animal models

Commonly used invertebrates include primates, cats, dogs, mice

Vertebrates have a advantage of greater homology with humans
than invertebrates, but huge disadvantages of practicality and
ethical concerns.
Mouse is the most commonly used mammalian model due to:

shorter generation time than other vertebrates

145

easier and less expensive to keep/maintain

easy to breed and select behaviour

commercial companies produce strains for purchase

used historically in top-down approach, used recently in

bottom-up approach

12.2.2 Choice of model

There are two different approaches to generating animal models to
investigate the genes responsible for a particular disease or trait:
the top-down and bottom-up approaches.

1) top-down approach
This approach focuses on isolating a particular behaviour then
screening the animals genes for variation that might contribute
to the behaviour.
Behaviour is isolated by artificially selecting animals to breed
based

on

behaviour,

which

establishes

two

strains

with

progressively greater differences in behavior.

Termed bidirectional selection

Examples of studies using the top-down approach:

1) Learning and memory: two strains of rats developed based on
their ability to learn how to pass through a maze

Error-prone rats bred with each other, error-free rats bred

with each other

Generated strains of dull rats and bright rats

2) Alcohol

preference:

multiple

mouse

strains

developed

demonstrating differences in alcohol preference, metabolism,

and withdrawal symptoms.

146

Alcohol preference measured as percentage of alcohol

consumed voluntarily by mouse strains compared to total
liquids consumed

Preferred percent of alcohol ranged from 1.7% to 9.4% in

four strains

Strains

investigated

for

differences

in

alcohol

metabolizing enzymes termed alcohol dehydrogenases

(ADH)
This

approach

advantageous

when

looking

for

genetic

differences within many genes or screening for novel unknown

genetic differences

1) Bottom-up approach
This approach focuses on mutagenesis to alter genes first, then
screening to identify mutations associated with behavioural
changes.
Two specific methods of generating genetically-altered animals:

Inserting a gene not usually present or increasing

expression of a gene not highly expressed

termed transgenic mice

Disrupting or turning off one or more genes through a

targeted mutation

termed knockout mice

Can have knockdown mice where expression is

just decreased

This approach advantageous when looking for behavioural

changes resulting from mutation of a specific gene or set of
genes thought to contribute to behaviour.

147

12.2.3 Choice of test

Once a model is selected, need to choose appropriate test to
measure animals behaviour.
The test must appear to measure requested criterion i.e.
construct and face validity
Higher function and human feeling can not be directly
modelled (eg low self-esteem, suicidal thought, depression)
Tests may instead measure comparable basic emotions and
comparable/homolog

fundamental

behavioural/neurochemical

mechanisms (anxiety, stress, memory)

Several behaviours can be assessed in mice using various tests,
for example:
1. Exploration vs Fear and Anxiety

Open-field test
Test

consists

of

recording

movements

and

behaviour when placed in an unfamiliar standard

environment
Count number of defecations to indicate anxiety,
locomotion to indicate exploration

Elevated Plus Maze or Zero Maze

Both mazes consist of a part open, part enclosed

maze

Montiors open area activity (time spent in open

areas and/or numbers of entries into open area);
more open area activity reflects anti-anxiety
behaviour.

Light/Dark Box

Consists of two compartments through which the

mouse can freely move, one darkened and one
148

illuminated

Test monitors amount of time spent in the

illuminated compartment;

Fearful animals prefer dark so more activity in

the illuminated area reflects anti-anxiety

2. Social Interaction

Placing two unfamiliar mice in a a free-movement

environment

Consists of placing the two unfamiliar mice in an

empty novel cage

Test monitors the type and number of interactions

between the mice, including active avoidance (one
mouse running away from the other), attacks or
aggressive behaviour, and grooming behaviour

Placing a free-moving mouse an three-chamber structured

environment

Consists of three chambers, first chamber with an

empty wire cup, an empty second chamber, and
third chamber with a wire cup containing an
unfamilar mouse.

Test monitors how much time/how many entries

the free-moving mouse makes into each chamber,
how many interactions with the unfamilar mouse

Low levels of social behaviour/interaction reflects

symptoms of several psychiatric disorders,
including autism, depression, or schizophrenia

3. Learning and Memory

Water maze or Morris test

Consists of placing a mouse in warm but opaque

149

water with a platform in a specific location.

Mouse aims to reach platform as quickly as
possible as mice dont like water. Initially the
platform is visible with cues on the wall during
training.

During testing, platform is placed under water (in

same location) and mice are tested for spatial
memory retention by recording time and distance
moved to locate the platform as well as time spent
in the target quadrant in relation to the 3 other
quadrants during the test session.

Longer times to reach the platform or less relative

time spent in the quandrant of platform indicates
poorer memory performance

Object recognition test

Consists of placing mouse in an empty open-field,

adding an object and habituating the mouse to its
presence, then removing the old object and
adding an identical old object and non-identical
new object.

Time spent with each object during training

sessions and test sessions are recorded with a
videotracking system (time spent with object is
defined as close investigation within 1cm of the
objects)

More time spent with the novel object indicates

recognition and memory of identical old object,
i.e. better memory performance

12.3 FAMILY/TWIN/ADOPTION

STUDIES

150

 Another

method

of

studying

human

behaviour

looks

at

inheritance of behaviours through families.

Can directly measure behaviours in humans (no need for
homologous behaviours as in animal models) but limited to
observational analysis as experimental intervention in human
families not ethical/possible.
Can look at inheritance through family trees (pedigrees), though
examination of parents and siblings, through examination of twin
siblings, and through examination of adopted siblings.

12.3.1 Family studies

Family tree studies / pedigrees were how inherited behaviour
was first studied historically

Sir Francis Galton published a study called Hereditary Genius

in 1869 looking at the number of eminent men among
families of eminent men compared to general population

Galton also looked at physical traits by whether physical

strength was more frequent in families of prominent Oarsmen
and Wrestlers.

Strong familial clustering suggests that the trait has a genetic

basis, families good for studying Mendelian traits, and families
are less susceptible to confounding heterogeneity i.e. same
ethnicity, similar environmental influences.

Disadvantages of family studies include difficulty separating of

shared environment and shared genetics, can be difficult to
obtain large-sized families for sufficient statistical power.

12.3.2 Twin studies

Galton developed twin studies.
After pedigrees, twin studies were used to study inheritance of
151

traits as this study design allows partitioning of the effects of

shared genes/environment.
Monozygotic (MZ) twins due to cleavage of the embryo at an
early stage

Formed from same egg and sperm which split after

fertilization

Thus

MZ

twins

share

nearly

100%

of

genetic

polymorphisms and correlations between MZ twins are

due to 100% shared genetic variance and common
environment: rMZ = VA + VC

Any phenotypic variation in behaviour and traits of MZ

twins are therefore due to non-shared environmental
causes; allows estimation of non-shared environmental
effects: VE = 1 - rMZ

Particularly useful in adoption studies where MZ twins

have been adopted into separate environments/families
from young ages

Dizygotic twins (DZ) due to polyovulation

Formed from two eggs released simultaneously fertilized

by two different sperm

Thus

DZ

twins

share

same

amount

of

genetic

polymorphisms as non-twin siblings (50%) and correlations

between DZ twins are due to 50% shared genetic variance
and common environment:

rDZ = 0.5 VA + VC

Comparing trait correlation in DZ compared with MZ

allows simple estimation of additive genetic variance and
common environmental variance:
= 2(rMZ rDZ)

and

VA

VC = rMZ VA

Complications of twin studies:

Environmental variance (VENV) can be common (VC) or

152

non-shared (VE) parenting / teaching can be non-shared

even if sibs are raised in same house or go to the same
school if each sib is treated differently

Non-additive genetic variance (dominance or epistasis)

complicates the estimation of genetic and environmental
variance and requires more complex analysis

Separating

VGEN

and

VENV

complicated

by

gene-

environment interactions (VGXE) and gene-environment

correlations (rGE)

DZ twins do share common pre-natal environment (in

utero)

MZ twins almost always share a chorion (part of foetal

membrane) while DZ twins almost always do not; this may
allow overestimation of genetic variance in MZ twins

12.4 CASE-CONTROL

ASSOCIATION STUDIES

More recently, case-control association studies used to study

inheritance of traits in unrelated individuals
Cases and controls should be matched on confounding factors
such as age, sex, ethnicity, and any other environmental factors
that may cause heterogeneity in the trait being studied
Can examine individual candidate genes, conduct genome-wide
association scans (GWAS), and most recently, exome or wholegenome sequencing.

12.5 PATHOLOGICAL

VARIATIONS IN BEHAVIOUR

Research into behavioural genetics has focused mostly on

pathological changes in behaviour, including Schizophrenia or
Alzheimers Disease.

12.5.1 GENETIC

STUDIES IN

SCHIZOPHRENIA

153

 Schizophrenia (SCZ) is a complex brain disorder affecting ~1

percent of the population.
Symptoms include delusions, paranoia, hallucinations, social
withdrawal, thought disorder, altered speech, catalepsy,
stereotypies, echopraxia and unusual posturing and mannerisms.
Divided into positive (excess of) and negative (loss of) symptoms
Twin studies analyzing concordance suggest a genetic
component
Main strategies for identifying SCZ genes include:

Linkage studies: Look for inheritance of common genes

in pedigrees with high number of affected people

Case-control association studies: look for genetic

differences in group of SCZ patients versus controls

Microarray expression analysis: Analyse differential

gene expression in autopsy brain tissue

Pharmacologic Animal Models: Drug induced state in

animal that mimics symptoms in humans, producing a
behaviour model which is useful for detecting
effectiveness of anti-schizophrenia medications i.e.
Amphetamine-induced stereotypy

Top-down approach Animal Models: Breeding of strains

that mimic schizophrenia symptoms in terms of
abnormal startle response (termed prepulse-inhibitiondeficit)

Bottom-up approach Animal Models: Dopamine

receptor knockout mice and glutamate receptor
knockdown mice

Animal models helped establish three of the most prominent

theories of schizophrenia: the dopamine hypothesis, serotonin
(or serotonindopamine) hypothesis, and glutamate hypothesis.

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Schizophrenia Dopamine Hypothesis

SCZ patients thought to have dopamine (DA) hypersensitivity
Dopaminergic system involved in locomotor activity, emotion,
motivation and cognition
Amphetamine causes excess DA release at the synapse
Can cause psychotic like symptoms in healthy people
Chlorpromazine (an anti-psychotic drug) blocks dopamine
receptor sites and relieves symptoms of SCZ in humans and
animal models
Dopamine receptor knock out mice have also been shown to
have reduced response to pharmacologically-induced SCZ-like
state compared to wildtype mice
Schizophrenia Serotonin Hypothesis
SCZ-like symptoms can be induced in pharmacologic animal
models and humans through hallucinogenic drugs (LSD,
mescalin) which mediate their effects via serotonin receptors.
Clozapine is effective in reducing the negative symptoms of
schizophrenia (e.g. disturbances of speech and flattening of
affect), which seems to work by blocking serotonin sites in the
brain.
Schizophrenia Glutamate Hypothesis
PCP and ketamine produce behaviour in healthy humans that
closely resembles symptoms of schizophrenia which works
through glutamate receptors.
Positive symptoms are paranoia, agitation, and auditory
hallucinations; negative symptoms include apathy, social
withdrawal and cognitive deficits, such as impaired attention
and working memory.
Glutamate receptor knockout or knockdown mice appear to
show abnormal locomotor patterns, prepulse inhibition deficit,
and poorer performance in spatial memory tests.
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12.5.1 GENETIC

STUDIES IN

ALZHEIMERS

DISEASE

Alzheimers disease (AD) is the most common form of dementia,

resulting in a decline or degeneration of cognitive functioning
from brain atrophy, leading to a wide range of symptoms.
Symptoms include confusion, aggression, mood swings, speech
problems, and memory loss.
Early onset AD (onset occurs between 30 to 60 years old)

also called familial AD (or FAD) due to direct

inheritance

rare form of AD = 5% AD cases

Late onset (onset occurs after 60 years old)

common form of AD

No definitive AD diagnosis except during autopsy. Medical

history, neurological evaluation (memory and mental tests etc)
can be used to diagnose AD as a cause of behavioural and
cognitive changes.
Brain atrophy can be assessed through imaging (MRI, PET
scan), and markers of degeneration may be tested for in
cerebrospinal fluid (CSF) analysis.
AD aetiology - cause unknown but most important risk factors
are age and family history (OR = 2.3), suggesting genetic
component.
There are many different theories for AD pathophysiology, but
two main hypotheses:

amyloid plaques - normal neuronal protein (amyloid

precursor protein) lyzed into -A fragments

Neurofibrillary Tangles (NFT) - normal microtubulestabilising Tau protein hyperphosphorylated,

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dissociates from microtubules

Bottom-up approach Animal Models developed to test role of
protein phosphatases (PPs) in AD as dysregulation of the PPs
may cause hyperphosphorylation of the Tau protein found in AD.

Protein phosphatases (PPs) remove phosphate groups

from proteins (opposite to phosphorylases)

PPs play a role in memory, are essential for synaptic

functioning

PP knockout mouse model produced, then tested for memory

task behavioural tests (spatial memory tested with water maze;
associative memory tested with object recognition task)

Water maze showed no difference between wildtype

and PP knockout mice in time spent in the platform
quadrant

Object recognition test showed a difference in

associative memory between wildtype and PP knockout
mice - recognition index greater for PP wildtype than
knockout, suggesting that PP function is important for
memory processes

12.6 NON-PATHOLOGICAL

VARIATIONS IN BEHAVIOUR

Non-pathological behaviours also widely studied in behavioural

genetics.
Study into non-pathological behaviours examines the genetic
causes of normal variation in certain behaviours.
Includes study into Intelligence, Anti-social behaviour
(Aggression, Irritability), and Personality traits
(Extraversion/Intraversion, Novelty-seeking or Reward-seeking
behaviours)

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12.6.1 GENETIC

STUDIES IN

INTELLIGENCE

Intelligence one of the first behavioural traits studied for

inheritance patterns: Galtons Hereditary Genius 1869

He compared the frequency of Judges of England,

Statesmen, English Premiers and other eminent men
among families of the well-known eminent men of his
time, compared to the general population

But Galton defined intelligence and success as having a

particular type of job; this is not an objective measure of
intelligence. In Galtons time surviving and flourishing as a
family over time required considerable effort.
Intelligence is defined now as: the ability to reason, plan, solve
problems, think abstractly, comprehend complex ideas, learn
quickly and learn from experience.
These things are more testable and measurable compared
with Galtons definition
Psychometric methods of measuring intelligence
Psychometric tests aim to measure intelligence in a standardized
way and have been found to have high statistical reliability, but
different tests measure intelligence differently
Main measure of general intelligence is Spearmans g factor

Spearmans g measures general intelligence

irrespective of subject matter (people with high values
of g will be reasonably successful in most subjects)

Correlated to large numbers of physiological

characteristics thought to be linked to intelligence

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High heritability estimates = 48 - 80%

Significantly associatied with the volume of gray matter

in the brain and higher g values correlated with a
higher volume of gray matter (r2 = 40%)

Also correlated with the following brain characteristics:

prefrontal lobe, overall brain mass, rate of brain
glucose metabolism, and cortical thickness

Other psychometric methods developed afterwards which test

intelligence in different ways and sometimes by separate
subjects or separate types of thinking/reasoning. These include:

StanfordBinet Intelligence Scales

Raven's Progressive Matrices

Scholastic Aptitude Tests (SAT)

Intelligence Quotient (IQ)

Each test has g-loading score which measures of general mental

ability (estimating Spearmans g)
Most widely-known and tested measure is IQ
Intelligence Quotient (IQ)
Calculated by setting median result from general population
sample to an arbitrary point on IQ scale (100 IQ points).
An individuals result from the test is ranked compared to the
results of normalization sample. For example, 1 standard
deviation in the population tested may equal 15 IQ points
IQ has been shown to be a quantitative trait with a normal
distribution in populations which is also highly heritable
IQ twin and adoptive studies
Different heritability (h2) and common environment analyses (c2)
have been done with IQ
MZ twins vs DZ twins reared together have h2 of 50-90%

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 MZ twins reared apart have h2 of 75%

DZ twins reared apart have h2 of 47%
Unrelated individuals reared together have c2 (estimation of
environmental component) equal to only 4%
Correlations between different sibs (twin, non-twin, adoptive)
show that shared environment doesnt contribute a lot of
variance in IQ. In other words, a shared environment but no
shared genes will give a low correlation in IQ scores.
The search for IQ genes
Can examine the genetic basis of diseases involving mental
retardation to identify genes that may be essential for
functioning intelligence, however many genetic syndromes
result in mental retardation
Some candidate genes identified through studies:

X-linked nonspecific mental retardation and GDI1

Microcephaly and ASPM

However, candidate gene approach failed to yield replicable

results (i.e. genes that are consistently associated with
intelligence in different populations).
Can also examine biological pathways which relate to normal
brain function (i.e. neurotransmitters)

APOE-4 and COMT have been marginally associated

with IQ

Can also conduct genome-wide scans (GWAS) for IQ genes

IQ Quantitative Trait Loci (QTL) Project in 2001 aimed
to identify QTLs of high IQ (>160) compared with
average IQ (~100) by examining 1842 simple sequence
repeat (SSR) markers spaced at 2 cM throughout the
genome
Found no significantly associated regions

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 A GWAS in 2008 scanned 500,000 SNPs from 7,089

children and found 6 SNPs associated with IQ
However, no polymorphisms were found in proteincoding regions and overall the variation in the 6
detected SNPs accounted for only 1% of the variance in
IQ problem called missing heritability

12.6.2 GENETIC

STUDIES IN

ANTI-SOCIAL

BEHAVIOUR

First studies into aggression and genetics in the late 1960s

looked at a cytogenetic abnormality - XYY males in hospitalized
and institutionalized men

First study in Scotland 1965 found 7 patients out of 203

(3.5%) to be XYY, compared to 0 out of 209 randomly
selected males (0%), but no statistical analysis

Falsely characterised them as aggressive and violent

based only on their presence in the hospital

More recently studies into anti-social behaviour look at genes

related to brain activity i.e. neurotransmitters
Anti-social behaviour is defined as: deliberate interpersonal
aggression or lack of consideration to others which may cause
damage to society; includes low-level nuisance, minor offenses,
serious violence, and criminal behaviour
Methods of measuring anti-social behaviour:
Multiple measures and scales used to monitor / rate anti-social
behaviour
Originally based on criminal or violent crime convictions
(looking at indiviudals already convicted of crimes)
Self-reported anti-social or violent acts which may or may not

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led to a conviction
Attitude questionnaires which are predictive of violent or
criminal behaviour
Different heritability and genetic associations will be found
depending on behaviour measures
Heritability estimates using twin and adoptive studies:
Different types of anti-social behaviour and different methods of
measuring anti-social behaviour can have widely different
heritability measures, especially if not controlling for GxE
interactions and correlations

Heritability estimates have ranged from h2 = 0 to 71%

Meta-analysis of 51 twin and adoption studies in 2002 showed

the following estimates of variance components:

VA = 32% (additive genetic effects)

VD = 9% (nonadditive genetic effects)

VC = 16% (common environmental effects)

VE = 43% (non-shared environmental effects)

The search for anti-social behaviour genes:

Many genes examined for association with anti-social behaviour,
most studies focus on polymorphisms in the following genes or
which may regulate the following genes:

COMT (cathechol-O-methyltransferase)

5-HTT (Serotonin transporter)

TPH (tryptophan hydroxylase)

DRD4 (dopamine D4 receptor gene)

MAOA (monoamine oxidase A) - metabolizes

neurotransmitters such as norepinephrine, serotonin,
and dompamine

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Key study in anti-social behaviour: Capsi et al. 2002 (Science

V297 p 851-854)
Seminal work by Capsi et al. 2002 examining both genetic
polymorphism in MAOA promoter and environmental factor of
maltreatment, as well as GxE interactions
Looked at four different measures of anti-social behaviour which
were shown to correlate well:

Diagnosed with Conduct Disorder according to

diagnostic criteria

Conviction for a violent crime by age 26

Psychological assessment using the Disposition Toward

Violence Scale at age 26

Peer assessment using Antisocial Personality Disorder

symptom scale (completed by "someone that knows
them well")

Childhood maltreatment shown to induce changes to

neurotransmitter systems; hypothesised that differences in
MAOA will handle these changes differently
Findings:

No maltreatment = both low and high MAOA

expressors had zero or negative index for antisocial
behaviour

Probable maltreatment = low and high MAOA

expressors had elevated but similar indexes for
antisocial behaviour

Severe maltreatment = both low and high MAOA

expressors had elevated index for antisocial behaviour
BUT low MAOA expressors had much larger index of
antisocial behaviour than high MAOA expressors

Conclusion: GxE effect of low MAOA expression and severe

maltreatment

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Other findings for link between MAOA and anti-social

behaviour:
Animal models - MAOA knockout and transgenic mice associated
with increased aggression
Association studies - null MAOA allele linked with antisocial
behaviour in males within a Dutch family

TOPIC 12: BEHAVIOURAL GENETICS PRACTICE QUESTIONS

1. List the important considerations when choosing an animal
model to study human behaviour.
2. Describe the type of approach to creating an animal model that
focuses on gene manipulation.
3. What kind of tests can be used to measure anxiety in mouse
models and what kind of validity should these tests have if they
are appliable to human behaviour?
4. True or False: Drosophila behaviour is not complex enough to be
a good model to study behaviour genetics.
5. Outline one of the methods that has been used to study a
pathological change in behaviour and what it has discovered
about the disease.
6. Describe two difficulties in studying the genetic basis of
intelligence or anti-social behaviour.
7. Why are neurotransmitter or neurotransmitter-related genes
studied most in relation to behaviour?
8. What is does GxE mean and why is it important in behavioural
research?

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End

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