Molecular Pathology –

(I) Gene mutation and level of protein.

A.M.R.Taylor
Institute of Cancer & Genomic Sciences
The effect of mutation on protein function?

 Most common effect is loss of function of protein

 Gain of function
 Acquisition of novel property
Expression at wrong time /wrong place
Loss of function

Due to gene deletion - leading to reduction in

gene dosage.

e.g. a-thalassaemia - deletion of a-globin gene.

e.g. ataxia telangiectasia – loss of ATM

Severity of disease that results from loss of
function mutations can generally be correlated
with amount of function lost - allelic heterogeneity

Retention of a small degree of residual function

greatly reduces severity of disease.
Gain of function mutation

Results in

 Increase in abundance of protein

 Increase in ability to perform one or more functions

e.g. achondroplasia - increase in normal function of
FGFR3 - (but has deleterious effect)
Receptors With Intrinsic Tyrosine Kinase Activity
FGF RECEPTOR
FGFR3 - transmembrane tyrosine kinase receptor that binds
fibroblast growth factors

Binding of FGF to extracellular domain activates

intracellular kinase domain - initiates signal cascade

In endochondral bone FGFR3 activation inhibits proliferation

of chondrocytes –slowing down formation of bone-
helps to co-ordinate growth.

 FGFR3 mutation increases its activity - gain of function

Cause FGF independent activation of tyrosine kinase domain

Causes inappropriate inhibition of chondrocyte proliferation

- leads to shortening of long bones - achondroplasia.
No allelic heterogeneity in achondroplasia

There is only one position where an amino

acid change in FGFR3 receptor can cause
achondroplasia - 1138 G>A at a CpG- (Gly380Arg).

 Gain of function mutations are likely to require a much

more specific change.

 Mutational homogeneity is a good indicator of gain of

function.
Gain of function mutation

Mutation that increases production of normal protein

Trisomy 21- Down syndrome
Multigenic traits
Simple example is digenic disorder

Eg. retinitis pigmentosum

Two rare mutations, each in a different unlinked
gene encoding two proteins in the photoreceptor.

Patients heterozygous for both peripherin and Rom1

develop the disease.
Real life

 People; mutations and their effect on

proteins.

 How can you the laboratory scientist

help?
Scenario
A clinician has a patient - a child with evidence of
neurodegeneration and may have the inherited
disorder ataxia telangiectasia.

The clinician wishes to know;

1. Does my patient have this particular rare disorder?

2. Are all patients, the same, with the disorder? Is my
patient different?
3. What else can you tell me about my patient? eg in
terms of prognosis.

I can send you a sample of blood.

 Ataxia telangiectasia
Autosomal recessive disorder.

 Early onset cerebellar ataxia (<2y)

Progressive cerebellar degeneration -
wheel chair by teenage
 Speech difficulties
 Abnormal eye movements
 Telangiectasia – dilated blood vessels
 Immune deficiency
 Large increased risk of malignant disease
- mainly lymphoid tumours in childhood.
 Increased chromosome instability
 Increased radiosensitivity
Median age of death ~ 18y
 Ataxia telangiectasia

Ocular telangiectasia
 Where do we start?

 What are the different laboratory approaches

that we can use to help with the diagnosis, and
obtaining further useful information?
 Use of different methods and reagents,
antibodies, westerns, immunofluorescence.
Ionizing radiation induced chromosome damage in a lymphocyte from a patient
with ataxia telangiectasia

Broken triradial chromosome

Chromatid type gap

Triradial chromosome

Chromatid type gap

Shapes of normal chromosome

Gamma - ray induced chromosome damage in
lymphocytes from a classical A-T patient

Following exposure to 1.0 Gray gamma-rays @ G2

No. tg tb
cells

Classical A-T 50 65 18
Normal control 50 8 0

tg = chromatid gaps
tb = chromatid breaks
Increased radiosensitivity of ataxia telangiectasia cells

Normal

Ataxia telangiectasia
 What is significance of increased radiation sensitivity?

 Meaning at the cellular level?

Radiosensitivity indicates an inability to repair DNA double strand breaks.
The function of the ATM protein is to protect our cells against DNA/
chromosome damage.
 Would you expect a carrier (heterozygote) to show
this increased radiosensitivity?
ATM protein in LCLs derived from A-T patients

Classical A-T

A-T
A-T
A-T
Normal

ME

PS
MK

GK

BM
RB
NK

CL
ZZ
ATM

Actin

1 2 3 4 5 6 7 8 9 10 11

Different levels of ATM protein

No ATM protein in patient with classical A-T
 Patients with ATM T7271G (Val2424Gly)
mutation- ATM protein expressed

Classical A--T
Normal

109II-1

109II-5

109II-6

136II-1
46II-2
ATM

Actin

ATM:Actin 13.5 0 17.2 18.6 14.4 7.5 14.8

(Patients with milder clinical features)

 What does the Western Blot tell us?

 Whether there is any protein present and how

much.
 This gives a clue about the type of mutations
present.
 If there is protein the Western gives no
information about whether it is still functional.
Total absence of ATM protein - interpretation

 Total absence of ATM protein is a feature of classical

A-T. The patient is likely to have A-T.

 If we take the finding of the Western at face value we

can also suggest that the two mutations in the patient
are null mutations and probably both truncating
mutations leading to instability (and loss) of the
protein.
Reduced level of ATM protein – interpretation

Depends:

Is it normal ATM?

Is it mutant ATM?

Might expect different effects

ATM protein in LCLs derived from A-T patients

Classical A-T

A-T
A-T
A-T
Normal

ME

PS
MK

GK

BM
RB
NK

CL
ZZ
ATM

Actin

1 2 3 4 5 6 7 8 9 10 11

Different levels of ATM protein

 Presence of normal levels of ATM protein – what does this
mean?

 The patient may not have ataxia telangiectasia. The protein is

in fact normal ATM protein present at normal levels.

 The patient may have A-T but expresses a normal level of

protein, but it is mutant protein. This might come about as the
result of the presence of two missense mutations.

If the protein is mutant its function should be affected.

 The patient may have A-T but caused by mutation.

in a different gene. So ATM would be normal!
 Where does the DNA sequence come from?

The ATM gene is ~100kb (100,000bases).

i.Sequence the individual coding exons. For this you need to know the
exon/intron structure. Where does one exon finish and the next one start.
There are 66 exons! The total coding sequence is just over 9000 bases.

ii.Coding sequence of course codes for mRNA (transcript), and so an

alternative for sequencing is to extract RNA from the cell and make copy
DNA (cDNA). This can then be sequenced. Only get sequence of transcript.
Could be different to Exon sequence. Could be useful.

What happens if the mutation destabilises the mRNA and so transcript is

produced?
Mutation analysis

i. Analysis in patients with no ATM protein.

 The expectation is that we would find two null

mutations.

 Two protein truncating mutations which result in

instability of the protein and total loss of protein.

 Null mutations can result from substitutions (may

introduce a stop codon TAA, TAG or TGA),
deletions and insertions (frame shift).

Re-read lecture 2
ATM splice site mutation, leading to loss of an exon-
instability and loss of protein
5763-1G>C

Intron Exon

Affected

Intron Exon

Mother

Intron Exon

Father
 Deletion giving rise to frameshift mutation, stop codon and
Genomic DNA instabilityGenomic
of truncated
DNA protein cDNA

Exon 10 (reverse) Exon 34 Intron 34 Exon 33 Exon 34

ATM truncating mutation

normal

Exon 10 (reverse) Exon 34 Intron 34 Exon 33 Exons 34 + 35

A-T210

c.1290_1291delTG mutation c.5177+5G>A mutation

causing frameshift causing exon 34 skip
Mutation analysis

ii. Analysis of mutations in patients expressing a

reduced level of protein.

 The expectation is that we would find at least one of

the two mutations with a missense mutation
(changing one amino acid to another).

 Possible that this protein retains some degree of

function.
Missense mutation resulting in protein expression
Forward

anisomycin treated

A-T 90

875 C T

Pro292Leu
 A-T 76

7660 C G

His2554Asp

A-T 88

8480 T G

Phe2827Cys
 Patients with ATM T7271G (Val2424Gly)
mutation - ATM protein expressed

Classical A--T
Normal

109II-1

109II-5

109II-6

136II-1
46II-2
ATM

Actin

ATM:Actin 13.5 0 17.2 18.6 14.4 7.5 14.8

(Patients with milder clinical features)

 A-T 46-3
7271 T G

A-T 46-4
7271 T G
 Problem
Chromosome 11

A B C D

Affected sibling Unaffected sibling

Why aren’t both siblings affected by this disorder -

they both inherit the same chromosomes from
mother and father
 Problem

Chromosome 11

A B C D

Affected sibling Unaffected sibling

Father not a carrier –

Affected son as a result of new mutation in paternal germ cell

 Maternally Derived Mutation

c.4588 G T p.(Glu1530Ter)

Father Mother A-T 23 Unaffected Daughter

Genomic DNA

No cDNA
available

cDNA
 Paternally Derived Mutation

c.8189 A C p.(Gln2730Pro)

A-T
Father Mother Affected Son Unaffected Daughter

Probably a de novo change during male meiosis

Mutations present should be consistent with
level of expressed protein

 No protein - then two truncating mutations

 Protein present - then either missense mutation

(may result in ‘normal’ level of protein)

(leaky splice site mutation

In frame exon deletion)