Download Renal transplant recipients

DNA Analysis
Dr Tony Fryer
Department of Clinical Biochemistry
& Centre for Cell and Molecular Medicine
North Staffordshire Hospital NHS Trust
& University of Keele
Overview
1. Background
2. Principles of DNA analysis
- Basic principles
- Techniques
3. New developments in technology
4. Novel applications - from single gene
disorders to high risk patient identification
5.Where is DNA analysis going in the clinical
laboratory?
1. Background
The current role of DNA-based tests
Generally used for:– single gene disorders
– small populations (rare diseases individually)
– patient diagnosis
But this restricted applicability is changing…...
Genetics revolution
• Increased public awareness
• Improvements in technology
• Greater understanding of genetic basis of disease
– Human genome project
• Increased interest from clinicians
• More requests for genetic tests
2.
Basic Principles of DNA analysis
DNA structure
• Double-stranded with 'sense' strand running in the
opposite direction to the 'antisense' strand.
• Strands connected by hydrogen bonding between
bases:
5’
A:T (2 bonds)
3’
C:G (3 bonds)
• Total number of bases in human sequence = 2.3 x 109
• Approx 50,000 genes.
3’
5’
Gene structure
• Exon
• Intron
5’
- encodes mRNA.
- between exons.
- spliced out during mRNA production.
• Promoter
- TAATA or Goldberg-Hogness Box.
- binding site for RNA polymerase.
- site of action of some hormone/receptors.
• CAT Box
- upstream control element (CCAAT Box).
- essential for accurate initiation of transcription.
• Enhancers - 5', 3' or intragenic.
- Regulate level of expression of genes.
• CAP site
- Transcription initiation point.
- caps mRNA - stabilises & ensures accurate translation.
• Poly A site - applies poly A tail to mRNA (stability & transport).
Mutation at any of these points can result in aberrant protein synthesis
3’
The Effect of Mutation
Normal base sequence:The man had one son and his dog was red but his son had one sad cat.
Substitution:The man had one son and his dog was red but his son hid one sad cat.
Deletion:The man had one son and hsd ogw asr edb uth iss onh ado nes adc at.
Insertion:The man had one son and his dog was red bus yth iss onh ado nes adc at.
Nonsense:The man had one son end.
Splice site mutations:The man had one wqt oen uts jfi pwx jei jsd pke zso nan dhi sdo gwa sre dbu thi
sso nha don esa dca t.
Trinucleotide repeats:The man had one son and his dog was red but but but but but but but but but but
his son had one sad cat.
Hybridisation (a)
• Concept central to the understanding of molecular biology.
• Relates to the hydrogen bonding between strands of DNA.
• Antisense strand = complementary to the sense strand:
5'-CCGGTCATTGCCAAGGT-3'
3'-GGCCAGTAACGGTTCCA-5'
• The two strands can be split (denatured) by heat and reanneal (hybridise) spontaneously when the temperature
drops below the melting temperature (Tm)
Tm depends on:1. Length of DNA sequence
2. Composition (GC:AT ratio)
Hybridisation (b)
• Under some circumstances (low stringency), non-identical
DNA sequences may hybridise:1. At lower temperatures
2. At high salt concentrations
• stringency determines specificity.
Restriction enzymes
• Naturally-occurring enzymes which cut DNA at specific
sequences (often palindromic)
Examples:
• EcoRI (Sticky ends)
5'-GAATTC-3'
5'-G
3'-CTTAAG-5'
3'-CTTAA
• SmaI (Blunt ends)
5'-CCCGGG-3'
5'-CCC
3'-GGGCCC-5'
3'-GGG
MboI
5'-GATC-3'
MstII
5'-CCTNAGG-3'
+
AATTC-3'
G-5'
+
GGG-3'
CCC-5'
Southern blotting (a)
• Digestion of DNA with
restriction enzyme
• Separation of fragments by
gel electrophoresis
• Transfer to a
nylon/nitrocellulaose
membrane
• Detection of sequence of
interest by a radio-labeled
probe
• Autoradiography
Southern blotting (b)
Mutation detection
• Mutation causes
loss/gain of
restriction site
• Fragment sizes
altered
• Different banding
patterns observed
(RFLP)
Southern blotting (c)
Disadvantages
• Labour intensive
• Expensive
• Use of radioactivity
• Not amenable to automation
• Not suitable for widespread clinical use
Polymerase chain reaction (a)
• Denaturation
ssDNA
• Annealing of primers
• Amplification
• 106 copies of a target
sequence
No of copies
• Repeat 25 cycles
No of cycles
Cyclin D1 gene
Exons: 1
2
3
4
5
3’
5’
C1722T
159 bp
PCR product
Hae III restriction site
20 bp
139 bp
Banding patterns following HaeIII restriction
CC
CT
TT
159 bp
139 bp
Cyclin D1 polymorphism
origin
159bp
139bp
Genotype
CT
CT
CT
CC
TT
CC
TT
CT
CC
CC markers
Polymerase chain reaction (b)
Advantages
• Uses v. small quantities of DNA
• Relatively cheap
• No requirement for autoradiography
• More amenable to automation
• Widespread clinical applications
Polymerase Chain Reaction
The start of a explosion in interest in
DNA technology:Single gene disorders are the tip of the
iceberg…..
Polymerase Chain Reaction
….but what lies beneath the surface?
What does the future hold?
PCR: the future
• Opening the door to new technology
• Opening the door to new applications
3. New developments in
technology
PCR - possibilities for automation
Stages in DNA analysis by PCR:
• DNA extraction
• Thermal cycling
• Product detection
PCR Automation - DNA Extraction
Options:
Capital cost Cost/sample Throughput
Phenol/Chloroform
Alkaline
Extraction kit
(e.g. Nucleon)
Automated system
low
low
£0.30
£0.15
10 samples/h
20 samples/h
low
high
£2
?£2
20 samples/h
100 samples/h
……but is extraction necessary?
PCR Automation - Thermal cycling
Scaling down
•
•
•
•
0.5ml tubes
0.2ml tubes
96/384 well plates
Capillaries (Light cycler)
Robotics
PCR Automation - Detection
Options
• Digest+Gel electrophoresis
• ARMS
• DASH – allele specific labeled probes
• Pyrosequencing – mini sequence analysis
• WAVE (Temperature Modulated Heteroduplex Analysis)
• Real-time PCR (e.g. Light cycler)
• Mass Specrometry
• Chip technology
Amplification Refractory Mutation System
(ARMS) - principle
mutant
Normal DNA
Normal DNA
normal
common
common
No amplifiction
PCR product
No PCR product
GSTM1 ARMS Assay
Exon 1
2
3 4 5
6
7
8
5’
3’
273 bp
132 bp
C/G substitution
273 bp
132 bp
110 bp
GSTM1 A
GSTM1 B
GSTM1 AB
GSTM1 null
GSTM1 ARMS gel
Amplification Refractory Mutation System
(ARMS) - advantages
• No requirement for restriction digestion
• Opportunities for multiplex analysis
– E.g. Elucigene CF20 kit
But…..
• Requires more Taq polymerase
• Still dependent on gel separation of PCR products
Automated gel-free detection systems
• Temperature gradient separation
– DASH
– WAVE
• Sequencing
– Pyrosequencing
Dynamic Allele Specific Hybridisation
• PCR
• Product immobilization
• Single strand isolation
• Probe hybridisation
• Read fluorescence while
heating
• Temperature-dependent
melting
• Analysis & allele scoring
Temperature modulated heteroduplex analysis
(WAVE)
•Useful for
screening for
unknown
mutations
•E.g. tumour
analysis
•More sensitive
/automated
than SSCP
Fragment separation by WAVE
The principle of pyrosequencing (a)
The principle of pyrosequencing (b)
4. Clinical applications
Classical Applications
Single Gene Disorders such as:
– Cystic Fibrosis
– Alpha-1-Antitrypsin Deficiency
– Haemochromatosis
Molecular diagnostics also applicable to:
– Tissue typing
– Viral infection
Cystic Fibrosis - background
• 'Single most common autosomal recessive disorder
among Caucasians.'
• 1:2500 live births
• Defective Gene:
- Cystic Fibrosis Transmembrane Conductance Regulator
(CFTR)
- Chloride Ion Channel
- Chromosome 7
- 250,000 base pairs
- 27 exons
- 1480 amino acids
CF: delta-F508 by site-directed
mutagenesis of PCR primers
Homozygous
negative
Heterozygous
carrier
Homozygous
positive
Heteroduplex
fragments
217bp
202bp
The delta-F508 mutation results in the loss of a
phenyalanine residue at amino acid 508
and accounts for around 80% of CF chromosomes
? Some CF gels in here?
Cystic Fibrosis - the classical single gene
disorder?
• Over 500 mutations in the CFTR now identified
• Mutation frequency depends on ethnic origin
• Demonstrates significant variation in phenotype:
Phenotype-Genotype Correlation
Genotype
% Pancreatic Insufficiency
F508/F508
99
F508/Other
72
Other/Other
36
• But even with the same causative mutation, phenotype differs
dramatically
• Do genetic factors predispose to severe disease even within
single gene disorders? - Modifier genes
Future Applications
• Pharmacogenetics
• Tumour analysis - oncogenes, TSG
• Detection of rearrangements - e.g. Philadelphia
chromosome
• Detection of residual disease
• Strain typing
• Chromosomal aberrations - FISH
• SNP analysis
– genetic predisposition to disease
– disease severity/prognosis (even in single gene disorders)
Renal transplant recipients a growing population
• World-wide increase in functioning transplants
– improved patient management - longer graft survival
– inproved access to transplantation
• Number of UK renal allograft recipients:
– 11,700 in 1994
– 18,400 in 1999
• Growing population who will develop
complications of long term immunosupression
Non-melanoma skin cancer a major complication
• Increased incidence
– 20-fold for basal cell carcinoma (BCC)
– 200-fold for squamous cell carcinoma (SCC)
• More aggressive behaviour
–
–
–
–
Present earlier
more numerous
grow more rapidly
metastisise earlier
• 5% of recipients will die as a consequence of these
maligancies
Can we predict which patients will
develop skin cancer within 5 years?
Will this affect patient management &
follow-up?
Clinical risk factors
• UV
–
–
–
–
–
–
–
–
–
Latitude
Outdoor occupation
Sunbathing habits
Cumulative sun exposure
Holidays abroad
Gender
Skin type 1
Blue or green eyes
Red/blonde hair color
• Immunosuppression
– Degree
– Regimen
– Duration
• Other
– Smoking (SCC)
– Premalignant lesions
– Arsenic exposure
1.00
Proportion tumor-free
AK negative
0.75
0.50
AK positive
0.25
0.00
0
10
20
30
Time from transplantation to appearance of first NMSC (years)
Genetic factors
•
•
•
•
•
UV-induced oxidative stress
Melanisation
Immune modulation
Detoxification of smoking-derived chemicals
Cell-cycle control
UV
Mn-SOD
EC-SOD
ROS
Immunomodulation
Lipid and DNA
hydroperoxides
GSTM1
GSTT1
GSTM3
GSTP1
TNF-
IL-10
TGF-
IFN-
Cyclin D1
Melanisation
Tyr
MC1R
VDR
Cell cycle
control
CYP2D6
Smoking
Gene-environment interactions
What effect does exposure have on associations
of GSTM1 null with skin cancer risk?
– GSTM1 null effect most evident in those with:
• High UV exposure (p=0.003, OR=11.5)
Tumour latency: Gene-Environment interactions
Proportion tumor free
1.00
0.75
Other genotype/sunbathing
score combinations
0.50
GSTM1 null+sunbathing score>3
0.25
0.00
0
5
10
15
Time post transplantation (years)
20
Targeted surveillance:
The predictive index
• Use stepwise logistic regression to obtain the best
set of predictors for developing NMSC within 5 or
10 years
• Generate a predictive index (score) that identifies
high risk patients
Predictive index (PI) - Australian model
PI = (K*1.23)+(A*0.085)+(S*1.47)+(M*0.62)-(G*1.15)-5.88
–
–
–
–
–
K= Actinic keratoses pre Tx; 1 if any present, 0 if absent
A = Age at transplantation
S = Skin type; 1 if type 1, 0 if types 2-4
M = Gender; 1 if male, 0 if female
G = GSTT1 genotype; 1 if null, 0 if A
If the score is -1.4 or greater, the model predicts a squamous cell
tumour within 5 years while if the score is less than -1.4, no
tumour is predicted.
•
•
•
•
Accuracy = 78.4%
Sensitivity = 82.0%
PPV = 46.3%
Specificity = 77.5%.
NPV = 94.8%
odds ratio = 15.7 (95% CI=7.7-31.9), p<0.0001.
Predictive index
- clinical application
These indices can be simplified and applied to
clinical management settings to:
– identify high risk patients for entry into clinical
surveillance programmes
– target appropriate treatments
– enable focusing of resources
– ?amend immunosuppresive dose
5. Where is DNA analysis going in
the clinical laboratory?
Clinical molecular genetics
- the future
• Will include very large numbers of patients
– every clinical speciality
• Includes areas other than just diagnosis
– management
– monitoring
– treatment
• Applicable to patients of every age (not just children)
Advances in technology will bring DNA analysis to the DGH
Molecular genetics - the future
Will the new applications provide sufficient
workload to warrant establishment of a new
Clinical Biochemistry sub-speciality?
A few final tips…..
1. Almost all DNA analyses require an
EDTA sample.
Cytogenetics require heparin.
If in doubt, request both!
2. Always ask for a family history and
ethnic origin of the patient