Molecular Pathology - (II)

Gene mutation and activity of protein.

A.M.R.Taylor,
Institute of Cancer & Genomic Sciences
 The problem
A clinician wishes to know whether his/her patient has
particular rare disorder.
He can send a sample of blood
Patient is a child : Evidence of neurodegeneration,
may have the inherited disorder : ataxia telangiectasia.
-*Ataxia telangiectasia is a recessive disorder. -Therefore,
affected individuals are either homozygous for the ATM mutation
or compound heterozygotes.
Distribution of ATM mutations in A-T cDNA

Mutations are mostly private (all different)

1 9168
 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
When is a DNA sequence change pathogenic?
 We consider deletions, nonsense mutations, frameshifts to be true
pathogenic mutations.
 Not every sequence variation is necessarily pathogenic
(polymorphisms). Any sequence change seen in more than 1% of
individuals may be a polymorphism.
 If the ‘polymorphism’ is rare in the population and is found in an A-T
patient how can we know whether it is pathogenic.
 Some clues can be gained by considering the actual sequence
change.
When is a DNA sequence change pathogenic? A missense mutation is more
likely to be pathogenic if it:

i. Affects a functionally important part of the protein.

ii. If the amino acid is conserved over evolution, it is more likely to be important.

iii. If amino acid substitutions are non-conservative (polar for non polar, acidic for
basic) they are more likely to be pathogenic.

This is an important problem – finding out how severe the

effects are of a missense mutation.
Algorithm of mutational nature of sequence change (Mutation
Taster)
Analysis comprises:
 Evolutionary conservation,
 Splice-site changes,
 Loss of protein features
 Changes that might affect the amount of mRNA.
 Test results are then evaluated by a naïve Bayes classifier 2,
which predicts the disease potential.
When is a DNA sequence change pathogenic? Really need a test
of the activity/function of the protein.
What can we learn from investigating the protein that is
present?
 Loss of function mutations
 Total loss of function results from total absence of the protein.
When a disorder results from total loss of function of a protein,
any mutation resulting in inactivation of the protein will give the
same clinical result.
 Total loss of function may occur even when some protein is
expressed Protein function.

 Where there is no protein produced of course there can be no activity.

 Some patients may have a less severe clinical phenotype; May be associated with a
milder mutation eg. a missense mutation producing some protein possibly with
residual activity.

 This activity can be assayed.

 The ATM protein

Putative Leucine SH3 Binding

PI-3 Kinase-Like Domain
Zipper Domain
1217 1238 1373 1382
p53 Binding ß-Adaptin Binding
Domain Domain
FAT domain
246 811 950 1966 2566 2711 2962
0
3056aa

9168 bp

ATM has kinase activity

 ATM kinase activity – we can measure this.

 Has to be induced by DNA damage

(protein level is not induced, just activity)

 Commonly use ionizing radiation or a

radiomimetic drug to activate

 Immunoprecipitate the ATM protein using an *ATM Ab* and

use it to phosphorylate a protein target (p53) in vitro
OR
 Use phosphospecific antibodies to detect the
phosphorylated target
 In vitro ATM kinase assay

Classical A-T
Classical A-T
Normal
Normal

A-T PC
Normal
-irradiation - + - + - + - +

ATM

GST-p53 (1-72) @ 1h

GST-p53 (1-72)
overnight
1 2 3 4 5 6 7 8
 Using phosphospecific antibodies

Classical

Classical
Normal
No ATM, No ATM activity

A-T 1

A-T 2
- + - + - + Irradiation (2Gy, 30min)

ATM Ser1981

ATM

Smc1 Ser966
Phosphospecific antibodies – Recognize
the phosphorylated protein – but not the
Smc1
unphosphorylated.
KAP-1 Ser824

KAP-1
Nbn Ser343

Nbn

CREB Ser121

CREB

1 2 3 4 5 6
 Some retained activity in three different mutant ATM proteins

c.5763-1050A>G

p.Phe2827Cys
A-T (classic)

p.Trp412Arg
c.8480T>G;

c.1234T>C;

Normal
Normal
-- + -- + - + - + - + - + irradiation
Advantage of using several
targets ATM Ser1981
A

B ATM

ATM overexposed

D Smc1 Ser966

E Smc1

F KAP-1 Ser824

G KAP-1

Nbn Ser343
H

Nbn
I

1 2 3 4 5 6 7 8 9 10 11 12
 Normal A-T 7184-1 7184-2 AT5
Mutant ATM with activity
- + - + - + - + - +

A ATM Ser1981

B ATM

ATM
C Overexposed

D Smc1 Ser966

E Smc1 c.7184C>T
p.Asp2395Val
F KAP-1 Ser824

G KAP-1

H Nbs1 Ser343

I Nbs1

J CREB Ser121

K CREB

1 2 3 4 5 6 7 8 9 10
 The activity of the V2424G (7271T>G) mutant protein

Low specific activity

p.Val2424Gly (1) (REF)

p.Pro1922fs (hom)

p.Pro1922fs (het (
Normal control

p.Val2424Gly (2)

p.Val2424Gly (3)
p.Gly2891Asp
leaky))
(leaky)

- + - + - + - + - + - + - + irradiation

ATM

Smc1 S966

KAP-1 S824

Nbs1 S343

Nbs1

1 2 3 4 5 6 7 8 9 10 11 12
 Activity changes over time

p53 ser15 phosphorylation time course for normal and classical A-T
patients

Normal A-T A-T

(classical)

Time after IR (h) 0 1 2 4 0 1 2 4 0 1 2 4

p53 ser15-P
Actin

Measuring activity of ATM

Other kinases phosphorylate targets at later times
 Band shift in Nbs 1 and hMre11
(phosphorylation by ATM )

Mllder patients

Class 109II-5 136II-1

Normal 118-3 109II-1 46II-2

Irradiation _ + _ + _ + _ + _ + _ +

Nbs1
P
hMre11

Measuring kinase activity of ATM

 Normal (M)
Activity of the ATM kinase in different cells

A-T (A)

B
J
- + - + - + - + - +

ATM Ser1981

ATM

Smc1Ser966

Smc1

Nbs1Ser343

Nbs1

p53 Ser15

p53
1 2 3 4 5 6 7 8 9 10
Possible consequences of mutant ATM protein
May have some retained function
- Could allow some degree of normal response to radiation
- Could affect level of radiosensitivity - measurable
- Gain of function
May result in altered interactions with other proteins
1. Relationship between level of ATM kinase activity and
function (preventing chromosome damage) of ATM
Increasing level of chromosome damage
ATM status (No. patients) Damage per cell
with decreasing ATM kinase activity*

No ATM (10) 1.91

With ATM but no activity (11) 2.05

With residual kinase activity (12) 1.17

With low level normal ATM (10) 0.35

Normal (14) 0.16

 2. ATM mutation causing cancer

FAMILY 109 FAMILY 46

I
1 2 1 2 3 4 5 6 7 8
82y 55y 50y 50y

II
1 2 3 4 5 6 1 2
50y 44y

III
1

= Indicates family members with breast cancer

= Indicates family members carrying the 7271T->G ATM mutation- high risk mutation
 Haploinsufficiency

For most gene products half the amount of protein (and it may be
a lot less) is sufficient for normal function.
For some gene products, however, 50% of the normal level is not
enough for normal function and haploinsufficiency produces an
abnormal phenotype.
Dominant negative effects

A non-functional mutant polypeptide can interfere

with a normal protein from the normal allele giving
a dominant negative effect.
Conclusions from molecular pathology –
indications for prognosis?

No protein expression at all –

Associated with classical disease.
Outlook for patient associated with classical prognosis.

Some protein expression.

May be associated with a less severe picture.
Difficult to suggest to a patient that this will mean a better
outlook, even if this is suggested by the presence of
residual protein function – matter of confidence.
Need a lot of data

Is there is quantitative relationship between protein

function and improved clinical outlook?

What about other interacting genes in the family.

 Modifying genes
 The same mutant allele can have different phenotypic effects on different
genetic backgrounds.

 This depends on particular alleles at other gene loci.

e.g. Less severe features of Beta-thalassemia in homozyotes who also inherit

an alpha-thalassemia
mutation - which acts as a modifier

Cystic fibrosis

Patients who are homozygous for the most common mutation

have highly variable lung disease.
Influence of modifying genes.

Ataxia telangiectasia

The degree of neurological manifestations are all independent of

each other - modifying genes
 Reliability & Independence of Scale Items

All items:
48 1.0
0.8
0.6
Correlate better with overall score
0.4
than with each other.
Contribute Independently to the
overall score.
50
Are reliable by intra- and inter-
50
observer tests.
Surviving Items do NOT
represent well the overall
functional state of an individual
54 55
with A-T Tom Crawford
- Generally start as circles
progress to spiky figures
- Significant inter-familial
56 58
variability (vertical axis)
- Appearance of intra-
familial similarity
(horizontal axis) Acknowledgment
to Tom Crawford
Intrafamilial Hetero/Homogeneity star plots of sibling pairs

Most startling evidence

for possible modifying
gene in A-T.

 No neurodegeneration even
when there is no ATM protein
present.

Locus
heterogeneity
 Same disease can be caused by
mutation in a different gene.
 Proteins interact with other proteins,
form complexes or are part of a
biochemical pathway. ** e.g. ataxia
telangiectasia can be caused by
mutation in the ATM gene but also
in the hMRE11 gene.
Absence of Mre11/ Rad50/NBN in A-T like disorder patient

Classical A-T
Normal

AOA2

ATLD
WL

CG
MT

TF

FP

JK

EF
JE
ATM (top band)

Senataxin

hRad50

Nbn

hMre11

Aprataxin

1 2 3 4 5 6 7 8 9 10 11 12
Absence of ATM kinase activity in MRE11 mutant patients

A-T (classical)
A-T (classical)
Normal

ATLD
- + - + - + - + irradiation
A ATM Ser1981

B ATM

C Smc1 Ser966

D Smc1

E KAP-1 Ser824 Mre11 is required for

full activation of ATM
F KAP-1

G Nbn Ser343

H Nbn

I CREB Ser121

J
CREB

1 2 3 4 5 6 7 8
 ATLD3 and ATLD4 - Mutations in hMRE11

A G 350 117 Asn Ser (Paternal) - missense

asparagine to serine.

C T 1714 572 Arg Ter (Maternal) - nonsense

 Null mutation in both alleles would be lethal evidence from mouse

models.
 Essential gene
Nonsense mediated mRNA decay
 Destabilisation of mRNA.

 It is not possible to obtain any sequence data from the cDNA

because the RNA transcript is unstable and is not produced.

 Therefore no cDNA can be synthesized.

 Maternal MRE11 mutation

1714 C T
A [-T] 4 A [-T] 5

Genomic
DNA

cDNA
(Maternal mutation
not seen)
ATLD3 and ATLD4 - Mutations in hMRE11

c.1714 C T p. (Arg572Ter)(Maternal) – nonsense, CGA (arginine) to TGA

(stop codon).
Nonsense mediated mRNA decay

 Using cells in culture

 Protect the RNA from decaying.
 Add an inhibitor protein synthesis.
 Prevents synthesis of the protein involved in destabilisation of mRNA.
 mRNA can now be extracted and cDNA made for sequencing.
The problem : A clinician wishes to know whether is/her patient has particular
rare disorder : He can send a sample of blood, Patient is a child, Evidence of
neurodegeneration. May have the inherited disorder ataxia telangiectasia. Of course it
frequently happens that the gene you thought was the cause of the disorder in your patient is actually
not the cause.
 What then?

 Other candidate genes

 It must be another gene, but which of the 25,000?

MRE11 c.1714C>T
You can take a ‘best candidate’ approach.

 Knowing the clinical picture of the patient - are there other genes involved in similar
functions or similar pathways which might be good candidates for the gene at fault.
 You then have to test these. How? Perhaps by using antibody on a Western to look
for a reduced amount of protein.
 Hope that you are right. Otherwise you will not find out the cause of the disorder.
 Quite a frequent happening.

Summary
Use different approaches to confirm the diagnosis.
 Analysis of protein expression;
 Analysis of protein activity;
 Identification of mutations.
 Should all be consistent with each other

Try and say something about the prognosis for the patient