Transcript Slide 1

DNA analysis
Molecular genetic testing for cystic fibrosis
Carolyn Tysoe
Principal Clinical Scientist
Royal Devon & Exeter NHS Foundation Trust
Outline
•DNA basics – structure, function, types of mutation
•Mutation detection
•Introduction to cystic fibrosis and CFTR gene
•CFTR mutations
•Testing strategy
•Case study
DNA transcription and translation
DNA
3’
mRNA
5’
G A GA A G T G C T T C T G C GA G
C U CUU C AC G AAG A CGCU C
nucleus
cytosol
Glu
Lys
Cys
Phe
Cys
Glu
G A GAA GT G C T T C T G C G A G
Glu
Lys
Cys
Phe
Cys
Glu
5’
3’
AAA
Effects of single base substitutions
Wild type
Nonsense
Missense
Glu
Met
ATG
Glu
Met
ATG
Glu
Met
ATG
Lys
Cys
Phe
Cys
Glu
AAG
TGC
TTC
TGC
GAG
Lys
Cys
Phe
Stop
AAG
TGC
TTC
TGA
Lys
Arg
Phe
Cys
Glu
AAG
CGC
TTC
TGC
GAG
Premature protein termination
Pathogenic?
Splice site mutations
AG
DNA
GT
AG
Normal spliced mRNA
DNA
Exon skipping
DNA
Intron inclusion
DNA
Use of a cryptic splice site
DNA
GT
AG
GT
Deletions and insertions
of one or a few base pairs…
Wild type
Frameshift
Glu
Met
ATG
Glu
Met
ATG
Lys
Cys
Phe
Cys
Glu
AAG
TGC
TTC
TGC
GAG
Lys
Cys
Phe
Ser
Arg
AAG
TGT
TCT
Cys
Ala
GCG
AGG
…to one or a few exons
Normal
Single exon
deletion
1
2
3
1
2
3
1
2
3
1
3
Look for known mutations eg OLA
Search for unknown mutations
eg sequencing
Mutation detection methods
Look for single or multi-exon deletions eg MLPA
Polymerase Chain Reaction (PCR)
•Primers can be fluorescently labelled – fragments separated by size and colour
•PCR primers have a common tail – use one primer to sequence all fragments
•Designed to work under the same conditions using MegaMix (mostly!)
•PCR setup on 96-well plate by Biomek robot
•Reagent lots recorded using 2D-barcoded tubes
Method depends on mutation
spectrum of gene
CFTR gene and cystic fibrosis
Cystic fibrosis
• What is the mode of inheritance?
• What is the incidence and carrier
frequency?
• Who does it affect?
• What is the disorder characterised by?
Cystic Fibrosis
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•
•
•
•
Autosomal recessive
Incidence 1: 2500
Affects children and young adults
Carrier frequency 1: 25
Production of viscous mucus  obstructs
ducts and glands  affects many organs
 multisystem disease
Cystic Fibrosis
• What are the major clinical features?
• Any additional features?
Major Clinical features
Lungs:
Obstructive pulmonary disease
Bacterial infection (Pseudomonas)
Pancreas:
Impaired exocrine pancreatic function
 Insufficient secretion of lipolytic and
proteolytic enzymes
 Malabsorption, steatorrhoea, failure
to thrive
Other clinical features
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•
•
•
•
•
•
Meconium ileus
Rectal prolapse
Obstructive jaudice
Nasal polyps
Sinusitis
Clubbing of fingers
Congenital bilateral absence of Vas Deferens
(CBAVD) in males
• Reduced fertility in females
The CFTR gene
•
•
•
•
•
•
Identified in 1989
Long arm chromosome 7 (7q31.2)
230kb of DNA
27 exons
6.1kb mRNA
1480 amino acids
Name:
Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR)
CFTR gene to protein
mRNA
CFTR mutations
• What is the mutation spectrum?
• What is the most common mutation?
CFTR mutations
42
10
16
2
13
0.5
15
1546 mutations listed on mutation database
www.genet.sickkids.on.ca/cftr
CFTR mutation spectrum
Mutation type
Missense
Frameshift
Splicing
Nonsense
In Frame ins/del
Large ins/del
Promoter
Polymorphisms
Frequency of
mutation type (%)
42
16
13
10
2
3
0.5
15
Most common CFTR mutation
p.Phe508del (F508)
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•
•
•
3bp deletion (CTT)
Deletes phenylalanine at codon 508
75% in UK population
66% in world population
Mutation classes
Increasing
severity in
phenotype
Variable splicing of exon 9
5T or 7T or 9T
GTGTG(T)AACAG
Exon
DNA 8
Exon 9
Intron 8
Exon10
Intron 9
Variable splicing of exon 9
Exon 8 Exon 9 Exon10
Exon 8 Exon10
Functional CFTR
Non-functional CFTR
9T
100%
0%
7T
90%
10%
5T
40%
60%
F508/R117H genotypes
F508/R117H
(7T)
CBAVD or
Pancreatic sufficient CF
F508/R117H (5T)
PS CF
Spectrum of CFTR disease
Estivill et al Nature Genetics 1996
CF testing at Exeter
• What referral reasons do we see?
• What molecular tests do we offer?
Referral reasons
• Establish or confirm the diagnosis of CF in symptomatic
individuals
• Failure to thrive
• Chronic cough
• Persistent chest infections
• For carrier detection in at-risk relatives and their
reproductive partners
• In prenatal testing of at-risk pregnancies and in which
foetal echogenic bowel has been identified
• Infertility investigations (CBAVD)
• Sperm and egg donor screening
Molecular testing at Exeter
CF1 – detection of p.Phe508del (F508) by sequencing exon 10
CF33 – detection of panel of 33 different mutations using the
Oligonucleotide ligation assay (OLA)
CF33 OLA
• Multiplex PCR
• Ligation
• Electrophoresis
• Genemapper analysis
OLA product has unique combination of
electrophoretic mobility and fluorescence and
permits identification of CFTR genotype
Multiplex of 15 PCR reactions
1
2 3
9
4
10
7 8
5 6a 6b
XXXX
Normal/Normal
XXXX
XXXX
Normal/Mutant
XXXXXX
XXXXXX
Mutant/Mutant
XXXXXX
11 12 13 14a
14b 15
G
C
C
G
G
C
T
A
A
T
T
A
18
19
16 17a 17b
Normal
Normal
Mutant
Mutant
20
21
22 23 24
Normal result
01. Patient1 Blue
1500
1000
500
S549
965
R553
1211
G551
V520
1348
1535
I507
1024
F508
Q493
1717-G
G542
G542
820
883
1398
1257
1257
01. Patient1 Green
1500
1000
500
R347
1412
3849+4 A
1665
W1282
R334
2195
1780
N1303
3849+10kb C
1078
1100
R1162
1226
1303
3659
1408
3905
1628
2244
01. Patient1 Yellow
1500
1000
500
A455
R117
Y122
1067
1259
1092
2789+5 G
621+1 G
1339
1071
2183 AA
1898+1 G
700
1071
711+1 G
1558
G85
1484
Heterozygous p.Phe508del
01. Patient1 Blue
1500
1000
500
S549
965
R553
1211
G551
V520
1348
1535
I507
1024
F508
Q493
1717-G
G542
G542
820
883
1398
1257
1257
F508
01. Patient1 Green
803
1500
1000
500
R347
1412
3849+4 A
1665
W1282
R334
2195
1780
N1303
3849+10kb C
1078
1100
R1162
1226
1303
3659
1408
3905
1628
2244
01. Patient1 Yellow
1500
1000
500
A455
R117
Y122
1067
1259
1092
2789+5 G
621+1 G
1339
1071
2183 AA
1898+1 G
700
1071
711+1 G
1558
G85
1484
Homozygous p.Phe508del
01. Patient1 Blue
1500
1000
500
S549
965
R553
1211
G551
V520
1348
1535
I507
Q493
1717-G
G542
G542
1024
883
1398
1257
1257
F508
01. Patient1 Green
1623
1500
1000
500
R347
1412
3849+4 A
1665
W1282
R334
2195
1780
N1303
3849+10kb C
1078
1100
R1162
1226
1303
3659
1408
3905
1628
2244
01. Patient1 Yellow
1500
1000
500
A455
R117
Y122
1067
1259
1092
2789+5 G
621+1 G
1339
1071
2183 AA
1898+1 G
700
1071
711+1 G
1558
G85
1484
Genetics White Paper 2003
“Our inheritance, our future”
Realising the potential of genetics in the NHS
“The NHS should lead the world in
taking maximum advantage of the
application of the new genetic
knowledge for the benefit of all
patients”
Investment in genetics
2003
£50 million funding including:
• £5.5M for gene therapy (including £2.5M for CF)
• £3.5M to train up to 90 scientists
• £18M capital to upgrade NHS genetics laboratories
As a result of this investment
By 2006, genetic test results should be available:
• Within 3 days for urgent samples (eg. Prenatal)
• Within 2 weeks where the potential mutation is known
• Within 8 weeks for unknown mutations in a large gene
All laboratories to secure accreditation with CPA or
equivalent within 18 months
Testing strategy – extended CFTR
analysis for SCOBEC network
£6 million to achieve:
Salisbury
Cambridge
Oxford
Cardiff
Exeter
Bristol
 Increased efficiency
 rationalisation of tests
 introduction of robotics
 new IT system
 Increased capacity
 White Paper reporting times
 CPA accreditation
 Integration of genetics in pathology
Modernisation of Exeter Lab
DNA extraction
PCR
Sequencing
streamlined
Sequence analysis
Reporting time data
• 3 days for urgent samples
S
C
C
E
• 10 days for known mutation
O
• 40 days for unknown mutations
B
100
80
60
Salisbury
Cambridge
Oxford
Cardiff
Exeter
Bristol
3 days
10 days
40 days
40
20
0
20042005
20052006
20062006200620062007 Qtr 2007 Qtr 2007 Qtr 2007 Qtr
1
2
3
4
• Testing strategy – extended
CFTR analysis for SCOBEC
network
Extended CFTR testing
1. Sequencing of entire gene (27 exons)
2. Dosage analysis
Partial or whole gene deletions are
not detected by sequencing
CTTCAAG
CTTCAAG
CTTCAAG
CTTAAG
CTTCAAG
• When you sequence an exon – how do you know how many
copies there are?
• Need a quantitative (dosage) test
CFTR deletions and duplications
44 reported out of 1546 CFTR mutations
(2.9%) (CF mutation database)
1
2 3
9 10
4
7 8
5 6a 6b
Deletion
Duplication
11 12 13 14a
18
16 17a 17b
14b 15
19
20
21
22 23 24
MLPA probes
PCR primer sequence X
FAM
PCR primer sequence Y
P
Stuffer sequence
24 bp sequence specific
probes
Annealing of probes
PCR primer sequence Y
PCR primer sequence X
Stuffer sequence
Ligation of probes
PCR primer sequence Y
PCR primer sequence X
Stuffer sequence
A ligase enzyme ligates the 2 probes together – Only
annealed probes will be ligated
Samples are heated to denature
the probe from the DNA
Probe amplification
The probe is amplified using the common primer pair
All the probes can be amplified using the
same primer pair and PCR conditions
MLPA Results - Electrophoresis
Normal Control
CFTR Duplication Exons 6b-10
MLPA Results – Spreadsheet analysis
Normal Control
Duplication Exons 6b-10 (Red)
Deletion (Blue)
MLPA Results – graphical display
Normal Control
Duplication Exons 6b-10
Whole gene deletion
Testing for CFTR mutations
1995 1996 1997
2004
CF4 CF12 CF31
CF33
CFTR entire gene
80% 85%
92.5%
~98% mutations
92.5%
2006
Patient KF
• 4 year old boy
• Chest infections
• Abnormal liver ultrasound
• ?Cystic fibrosis
Patient KF
Routine test for CF Genotype
KF is heterozygous for p.Phe508del
Heterozygous p.Phe508del mutation in KF
Patient KF
Request for extended CFTR testing
• Referral criteria for extended
testing
Guidelines for extended CFTR testing
(Sequencing and MLPA £700)
a)
Obligate Carrier (Clinical diagnosis of CF in offspring)
b)
Risk factors
AND
AND
≥ 1 phenotypic symptom
(recurrent chest
infections, pancreatic
insufficiency or CBAVD)
And/Or
+ve NBS (two high IRT’s)
And/Or
CF diagnosed in a sibling
+ve sweat test or two
borderline sweat tests
And/Or
-/- or +/On 29 – 33 panel
mutation screens.
+ve NPD
If unable to obtain sufficient sweat or NPD
unavailable, proceed if ≥ 2 phenotypic
symptoms or ≥ 2 of the and/or risk factors
Patient KF
Request for extended CFTR testing
MLPA Result for KF
Duplication Exons 6b-10
KF
Patient KF
Sequencing identified a heterozygous
p.Glu585X mutation in KF
Heterozygous p.Glu585X mutation in KF
Results to date
Clinical diagnosis CF
Obligate carrier
Splice site/N
p.Phe508del/splice site (2)
c.1766+1G>A/splice site
p.Phe508del/truncating (3)
p.Phe508del/missense (3)
c.3067_3072del6 + p.Ile148Thr/missense
c.3717+10kbC>T/missense
p.Arg1070Gln/N
?CF
N/N (bronchiectasis x2)
p.Phe508del/N (bronchiectasis)
p.Ile618Asn/nonsense
N/N (Pancreatic sufficient)
Overall pick-up rate 13/18 (72%)
Pick up rate 13/15 (87%) excluding bronchiectasis
Summary
2 mutations
1 mutation
0 mutations
90%
92.5%
98%
81%
18%
1%
85%
14%
1%
96%
4%
<1%
 The OLA assay detects 33 CFTR mutations (predict 2
mutations identified in 85% of patients with CF)
 Preliminary data suggests that sequencing/MLPA increases
detection of 2 mutations to ~96% of patients
Therefore it is not possible to exclude a diagnosis of CF