Molecular basis of

Genetic diseases
Dr Tao Wang
1.014 A V Hill Building
Wednesday 26th 2011, 10:00
E-mail: tao.wang@manchester.ac.uk

Molecular Basis
of Genetic Diseases

Types of mutations affecting genes

Epigenetic changes affecting genes

e.g., mutation at regulatory region of a gene,

mutation affecting the protein product, frame
shift, splicing, etc.
Independent of DNA sequence change

Determinants of phenotypic expression

Genotype-phenotype correlation

Basic types of mutations

Mis-sense mutation

Nonsense mutation

Single nucleotide change introduce a stop cordon

Example: TGC (Cys)
TGA (STOP)

Synonymous substitution

Single nucleotide change leads to amino acid change

Example: CGC (Arg)
TGC (Cys)

No amino acid change

Example: CGC (Arg)

CGA (Arg)

Deletions, insertions
Trinucleotide expansion

CAGCAGCAGCAGCAGCAGCAGCAG

Over view of

Transcription and translation

Gene variations affecting any of the above process can give rise to disease phenotype by
generating abnormal polypeptides/proteins quantitatively or qualitatively.

How can a mutation cause

the clinical phenotype?

Loss-of-function mutation

Gain-of-function mutation

A mutation that results in reduced or abolished protein function

e.g., thalassemia, Duchenne muscular dystrophy, fragile X
syndrome
A mutation that results in an abnormal activity on a protein
e.g., Huntingtons disease, T-ALL

Dominant negative effect

Mutant product interfere with the function of the normal product in

a heterozygote
A special case of loss-of-function
e.g., COL1A1 or COL1A2 mutation

Loss-of-function mutation (I)

-- Regulatory Mutations

Haemophilia B (OMIM 306900):

Promoter mutations in human factor IX gene
Factor IX promoter region has:
Binding sites for transcription factors
LF-A1/HNF4 and C/EBP
- Mutations in this region cause
Haemophilia B Leyden
Androgen response element (ARE)
- Hormonally regulated
- Amelioration effect at puberty
- Haemophilia B Brandenburg
causes lifelong haemophilia B
Bowen D J Mol Path 2002;55:1-18
2002 by BMJ Publishing Group Ltd and Association of Clinical Pathologists

Loss-of-function mutation (II)

-- Protein Products - Quantitative
Thalassemia (OMIM 613985)

Autosomal recessive
Point mutation or deletion results in reduced rate of synthesis or no
synthesis of one of the globin chains that make up haemoglobin.
Too few globins synthesized

Developmental expression of globin chains

a2 a1

Gg

Ag

from embryo to adult

Hemoglobins:
Embryonic:

Fetal:
Adult :

z2e2 , a2e2, z2g2

a2g2 (HbF)
a2d2 (HbA2)
a2b2 (HbA)

Loss-of-function mutation (III)

-- Protein Products - Qualitative
Sickle cell disease
(OMIM 603903)

Autosomal recessive
Red blood cells abnormal, rigid,
sickle shape, decreases cells'
flexibility
Mutation: single nucleotide change
in the -globin gene

GAG (glutamic acid) to GTG (valine)

Abnormal function of globin

A qualitative problem

Loss-of-function mutation (IV)

-- Large deletions

Two typical conditions:

Duchenne muscular dystrophy (DMD)

Becker muscular dystrophy (BMD)

Caused by mutation in dystrophin gene (DMD)

Introduce Frame shift

Duchenne muscular dystrophy

(DMD) (OMIM 310200)

X-linked recessive; female carriers usually OK

Affects ~1 in 3,000 boys
Clinical:

Mainly affecting young boys (3-5 years)

progressive muscle weakness
OK up to age 10, wheelchair in teens, steady decline, die
in 20s (respiratory and cardiac involvement)

No effective treatment.

Becker muscular dystrophy

(BMD) (OMIM 300376)

X-linked recessive
Affects ~1 in 20,000 men
Same clinical picture as Duchenne
dystrophy, but symptoms very variable
and occurs much later
Can have normal lifespan

Dystrophin anchors the contractile machinery

to the sarcolemma

Burton & Davies Cell 108 : 5-8; 2002

Duchenne vs. Becker dystrophy

deletions in DMD, mis-sense mutations in BMD?
65% deletions in both DMD and BMD

large deletions in DMD, small ones in BMD?

no correlation with size of deletion

deletion of an essential part of the gene in DMD, other

parts in BMD?
deletions overlap;
in some cases a BMD deletion encompasses 1 or more DMD deletions

DNA

CATCATCAT

mRNA

protein

CATCATCA

3
TCATCATCAT

4
CATCATCAT

CATCATCATCATCATCATCATCATCATCATCATCAT

His - His - His - His - His - His - His - His - His - His - His - His

----------- --------------- Delete exon 2 ---------------- ----------DNA

mRNA

protein

1
CATCATCAT

3
TCATCATCAT

CATCATCATTCATCATCATCATCATCAT

4
CATCATCAT

Frame shift

His - His - His - Ser - Ser - Ser - Ser - Ser - Ser

DNA

CATCATCAT

mRNA

protein

CATCATCA

TCATCATCAT

4
CATCATCAT

CATCATCATCATCATCATCATCATCATCATCATCAT

His - His - His - His - His - His - His - His - His - His - His - His

-------------------------- Delete exon 3 --------------------------DNA

mRNA
protein

1
CATCATCAT

2
CATCATCA

4
CATCATCAT

CATCATCATCATCATCACATCATCAT.

His - His - His - His - His - His - Ile - Ile -

Frame shift

DNA

CATCATCAT

mRNA

protein

CATCATCA

TCATCATCAT

CATCATCAT

CATCATCATCATCATCATCATCATCATCATCATCAT

His - His - His - His - His - His - His - His - His - His - His - His

----------------------- Delete exons 2 and 3 ---------------------DNA

mRNA

protein

1
CATCATCAT

4
CATCATCAT

CATCATCATCATCATCAT

His - His - His - His - His - His

In frame

Dystrophin gene deletions

Gene sequence is 99.3% intron - so most deletion breakpoints
are in introns & remove one or more complete exons
Deletions of exons that
create a frameshift completely

Deletions that leave the

reading frame intact result in

abolish synthesis of dystrophin

in males

synthesis of a smaller but

partially functional protein

consequence is

Duchenne dystrophy

consequence is

Becker dystrophy

Gain-of-function mutation

T-cell acute lymphoblastic leukemia (T-ALL)

(OMIM 190198)

NOTCH1 mutation in over 50% of patients

Activating mutations

Gain-of-function
Notch1 mutations in T-ALL

Two mutational hot spots are present within the extracellular heterodimerization
Domain (HD) and the C-terminal PEST domain.
HD mutations cause ligand-independent generation of activated Notch1 (ICN1);
PEST mutations sustain ICN1 activity.

Annual Reviews

Trinucleotide Repeats Expansion

Two examples:
Huntingtons disease

Gain-of-function mutation

Fragile X syndrome

Loss-of-function mutation

Huntington Disease (OMIM 143100)

Autosomal dominant
Late onset (typically 40s) Neurodegenerative
disorder
Involuntary movements
Memory loss
Apathy
Changes in personality and mood
depression and anxiety, Seizures

Molecular basis of HD
Genotype-phenotype correlations:
QQQQQQQQQQ

(CAG)n

>60 Juvenile HD
>37 pathological mutation
29-35 premutation
8-29 normal
Huntingtin gene
Exon 1

Molecular mechanisms involved

in pathogenesis of PolyQ diseases
Using HD as an example:

Everett C M , Wood N W Brain 2004;127:2385-2405

Fragile-X Syndrome (OMIM 309550)

Borderline to severe mental retardation

~20% autistic
Characteristic long face, large ears in adult
men
Macro orchidism in 80-90%
Incidence ca. 1 in 4,000 males

The Fragile-X chromosome

Marker X seen in a proportion of cells under special

culture conditions

Molecular basis of Fragile-X

(CGG)n

>230 full mutation

60-230 premutation

NKS

5UTR

NES

7-60 normal

FMR1 gene

RGG

3UTR

Function of the FMR1 gene

Wild
CGGtype
expended
FMRP function:
FMRP:

AAAAA

AAAAA

AAAAA

AAAAA

FMRP with a point mutation (1304N):

AAAAA

AAAAA

How does mutant FMRP1 leads

to Fragile-X?
Repeat is in 5 untranslated region of FMR1
Full expansion triggers methylation of the
DNA through histone deacetylation
Methylation switches off expression of the
gene
Loss of FMRP1 is the underlying mechanism
of Fragile-X Syndrome.

Unique properties of dynamic

mutations

Anticipation

A phenomenon whereby the symptoms of a

genetic disorder become apparent at an earlier
age and severer as it is passed on to the next
generation.
Repeat unstable, tend to expend
The size of expansion is often correlated with the
severity of symptoms and/or with onset at
younger age

Dominant negative effect

Osteogenesis imperfecta Type III (OMIM 259420):

Mutations in COL1A1 or COL1A2 genes

Bones fracture easily, deformity, Loose joints
Respiratory problems
Short stature, spinal curvature and sometimes barrelshaped rib cage
Poor muscle tone in arms and legs
Discolouration of the sclera
Early loss of hearing possible

Self-assembly of collagen fibres

Mutations with the most drastic effect on protein synthesis are not the one that
produce the most severe phenotypes.
The mutant protein has dominant negative effect on the triple helix assembly.

Molecular Basis
of Genetic Diseases

Types of mutations affecting genes

Epigenetic changes affecting genes
Determinants of phenotypic expression

Epigenetic Changes
Affecting Gene Expression

Heritable (cell to daughter cell, or through a pedigree )

changes in the gene expression that do not depend on
a DNA sequence change.
Mechanisms:

DNA methylation
Histone modification and Chromatin structure
RNA silencing by non-coding RNAs

Examples:

Genomic imprinting: AS, PWS

X-chromosome inactivation

Chromatin structure
- open vs closed

Berger Curr Opin Genet Dev 12 142-148 ; 2002

Angelman syndrome (AS)

(OMIM 105830)

slow development
unusual movements
sever learning difficulties
epilepsy
happy face (Happy Puppet
Syndrome)
poor communication skills and
little or absent speech

Prader-Willi syndrome (PWS)

(OMIM 176270)

marked hypotonia: low muscle tone

short stature excessive appetite
obesity
immature physical development
learning disabilities emotional
instability
almond shaped eyes with thin,
down-turned lips
nearly always left-handed

Both cases had Deletion of proximal

portion of chromosome 15 (15q11)

FISH (Fluorescent In-Situ Hybridization)

test for deletions

15q11 deletions

Paternal deletion

Maternal deletion

Prader-Willi

Angelman

Reason: Genomic imprinting

A phenomenon that gene expression of a small number of genes (~80) in mammals

depends on parental origin.

Molecular Basis
of Genetic Diseases

Types of mutations affecting genes

Epigenetic changes affecting genes
Determinants of phenotypic expression

Determinants
of phenotypic expression

Nature of the mutation

Mutation type
Mutation position

Genetic background

Different mutations in the same gene can lead to more- or less-severe

phenotypes depending on the effects of the mutations on the expression of
the gene or on the function of the protein product

Heterogeneity in genetic background

Environmental influences

Lifestyle, diet, and living environment may affect the disease

phenotype

Genetic background

The background genes may influence the disease

phenotype

Two individuals within a family may have the same mutated gene,
however, they will certainly (unless they are identical twins) have a
lot of genes that are not similar

Genetic variants (polymorphisms) in the same gene

may influence the disease phenotype

Example: polymorphism H558R has mutation-specific effects on

SCN5A-related Sick Sinus Syndrome (SSS)

Polymorphism modulate
disease phenotype
Whole-cell current recordings by patch clamping:

Loss-of-function defect of D1275N in SCN5A was rescued by

R558 through enhancing cell surface targeting and
improving steady-state activation of the mutant channels.
J Gui et al, J Cardiovasc Electrophysiol. 2010,;21(5):564-73.

Summary

Types of mutation affecting gene function

Los-of-function mutation

Gain-of-function mutation

Osteogenesis imperfecta Type III

Epigenetic mechanisms in genetic diseases

Huntingtons disease, T-ALL

Dominant negative effect

Thalassemia, Duchenne muscular dystrophy, fragile X syndrome

PWS, AS

Genotype-phenotype correlations