DNA analysis Molecular genetic testing for cystic fibrosis Carolyn Tysoe Principal Clinical...
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Transcript of DNA analysis Molecular genetic testing for cystic fibrosis Carolyn Tysoe Principal Clinical...
DNA analysisMolecular 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
ACA T TG G AA G G T C T CG G G5’3’DNA
nucleus
mRNA
G A G
Glu
G A G
Glu
T G C
Cys
T G C
Cys
T T C
Phe
A A G
Lys
DNA transcription and translation
cytosol
3’5’C U C GU U C A C G AA G A C C U C
AAA
Glu Lys Cys Phe Cys Glu
Effects of single base substitutions
Wild type Glu Lys Cys Phe CysMet Lys Cys Phe Cys Glu
ATG AAG TGC TTC TGC GAG
Missense Glu Lys Phe CysMet Lys Phe Cys Glu
ATG AAG CGC TTC TGC GAG
Arg Pathogenic?
Glu Lys Cys PheMet Lys Cys Phe
ATG AAG TGC TTC TGA
Nonsense Premature protein terminationStop
Splice site mutations
DNAGT GT GTAGAG AG
DNANormal spliced mRNA
DNA
DNA
Exon skipping
Intron inclusion
DNAUse of a cryptic splice site
Deletions and insertions
Wild type Glu Lys Cys Phe CysMet Lys Cys Phe Cys Glu
ATG AAG TGC TTC TGC GAG
Frameshift Glu Lys Cys Phe CysMet Lys Cys
ATG AAG TGT TCT GCG AGG
Ser Ala Arg
of one or a few base pairs…
1 2 3
1 3
Single exon deletion
1 2 3
1 2 3Normal
…to one or a few exons
Mutation detection methods
Look for known mutations eg OLA Search for unknown mutationseg sequencing
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
• 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
• 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
1546 mutations listed on mutation database
www.genet.sickkids.on.ca/cftr
4210162130.515
CFTR mutation spectrum
Mutation type Frequency of mutation type (%)
Missense 42
Frameshift 16
Splicing 13
Nonsense 10
In Frame ins/del 2
Large ins/del 3
Promoter 0.5
Polymorphisms 15
Most common CFTR mutation
p.Phe508del (F508)
• 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
DNAExon 8 Exon10
Intron 8 Intron 9
GTGTG(T)AACAG
5T or 7T or 9T
Exon 9
Variable splicing of exon 9
Exon 8 Exon 9 Exon10
Functional CFTR
Exon 8 Exon10
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 ExeterCF1 – detection of p.Phe508del (F508) by sequencing exon 10
CF33 – detection of panel of 33 different mutations using the Oligonucleotide ligation assay (OLA)
OLA product has unique combination of electrophoretic mobility and fluorescence and permits identification of CFTR genotype
CF33 OLA• Multiplex PCR• Ligation• Electrophoresis• Genemapper analysis
14b 15
1 2 3 4
9 10 11 12 13 14a
16 17a 17b
18 19 20 21 22 23 24
Multiplex of 15 PCR reactions
5 6a 6b
GXXXX
GXXXX
AXXXXXX
AXXXXXX
7 8
Mutant
Normal
MutantNormal
CC
Normal/Normal
AXXXXXX
GXXXXCT
Normal/Mutant
TTMutant/Mutant
1500
1000
500
R347
1412
3849+4 A
1665
W1282
2195
R334
1780
3905
1628 1078
1226
3849+10kb C
1100 R1162
2244
N1303
1303
3659
1408
S549
965
R553
1211
G551
1348
V520
1535
I507
1024
F508
820
Q493
883
1717-G
1398
G542
1257
G542
1257
1500
1000
500
A455
1067
R117
1259
Y122
1092 2183 AA
700
2789+5 G
1071
1898+1 G
1071
621+1 G
1339
711+1 G
1558
G85
1484
1500
1000
500
01. Patient1 Green
01. Patient1 Yellow
01. Patient1 Blue
Normal result
1500
1000
500
R347
1412
3849+4 A
1665
W1282
2195
R334
1780
3905
1628 1078
1226
3849+10kb C
1100 R1162
2244
N1303
1303
3659
1408
S549
965
R553
1211
G551
1348
V520
1535
I507
1024
F508
820
Q493
883
1717-G
1398
G542
1257
G542
1257
1500
1000
500
A455
1067
R117
1259
Y122
1092 2183 AA
700
2789+5 G
1071
1898+1 G
1071
621+1 G
1339
711+1 G
1558
G85
1484
1500
1000
500
01. Patient1 Green
01. Patient1 Yellow
01. Patient1 Blue
F508
803
Heterozygous p.Phe508del
1500
1000
500
R347
1412
3849+4 A
1665
W1282
2195
R334
1780
3905
1628 1078
1226
3849+10kb C
1100 R1162
2244
N1303
1303
3659
1408
S549
965
R553
1211
G551
1348
V520
1535
I507
1024
Q493
883
1717-G
1398
G542
1257
G542
1257
1500
1000
500
A455
1067
R117
1259
Y122
1092 2183 AA
700
2789+5 G
1071
1898+1 G
1071
621+1 G
1339
711+1 G
1558
G85
1484
1500
1000
500
01. Patient1 Green
01. Patient1 Yellow
01. Patient1 Blue
F508
1623
Homozygous p.Phe508del
“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”
Genetics White Paper 2003
Investment in genetics2003
£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
Salisbury
Exeter
Cardiff
Bristol
Oxford
Cambridge
Increased efficiency rationalisation of tests introduction of robotics new IT system
Increased capacity White Paper reporting times CPA accreditation Integration of genetics in pathology
£6 million to achieve:
DNA extraction PCR Sequencing Sequence analysis
streamlined
Modernisation of Exeter Lab
Reporting time data
C
EB
OC
S• 3 days for urgent samples
• 10 days for known mutation
• 40 days for unknown mutations
Salisbury
Exeter
Cardiff
Bristol
Oxford
Cambridge
0
20
40
60
80
100
2004-2005
2005-2006
2006-2007 Qtr
1
2006-2007 Qtr
2
2006-2007 Qtr
3
2006-2007 Qtr
4
3 days
10 days
40 days
• Testing strategy – extended
CFTR analysis for SCOBEC
network
Extended CFTR testing
2. Dosage analysis
1. Sequencing of entire gene (27 exons)
CTTCAAG
CTTCAAG
CTTCAAG
CTTAAG
CTTCAAG
• When you sequence an exon – how do you know how many copies there are?
• Need a quantitative (dosage) test
Partial or whole gene deletions are not detected by sequencing
CFTR deletions and duplications
44 reported out of 1546 CFTR mutations (2.9%) (CF mutation database)
1 2 3 4
5 6a 6b
7 8
9 10 11 12 13 14a
14b 15
16 17a 17b
18 19 20 21 22 23 24
DeletionDuplication
MLPA probes
PCR primer sequence Y
24 bp sequence specific probes
FAM
PCR primer sequence X
Stuffer sequence
P
Annealing of probes
PCR primer sequence YPCR primer sequence X
Stuffer sequence
Ligation of probes
PCR primer sequence Y
A ligase enzyme ligates the 2 probes together – Only annealed probes will be ligated
PCR primer sequence X
Stuffer sequence
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
Normal Control
CFTR Duplication Exons 6b-10
MLPA Results - Electrophoresis
Normal Control
Duplication Exons 6b-10 (Red)
Deletion (Blue)
MLPA Results – Spreadsheet analysis
Normal Control
Duplication Exons 6b-10
Whole gene deletion
MLPA Results – graphical display
Testing for CFTR mutations
1995 1996 1997 2004 2006
CF4 CF12 CF31 CF33 CFTR entire gene
80% 85% 92.5% 92.5% ~98% mutations
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)
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
+ve NPD
-/- or +/-On 29 – 33 panel mutation screens.
If unable to obtain sufficient sweat or NPD unavailable, proceed if ≥ 2 phenotypic symptoms or ≥ 2 of the and/or risk factors
Obligate Carrier (Clinical diagnosis of CF in offspring)a)
b)
Patient KF
Request for extended CFTR testing
MLPA Result for KF
Duplication Exons 6b-10
Patient KF
KF
Sequencing identified a heterozygous p.Glu585X mutation in KF
Heterozygous p.Glu585X mutation in KF
Results to date
Clinical diagnosis CF Obligate carrier
p.Phe508del/splice site (2)c.1766+1G>A/splice site p.Phe508del/truncating (3)p.Phe508del/missense (3)c.3067_3072del6 + p.Ile148Thr/missensec.3717+10kbC>T/missensep.Arg1070Gln/N
p.Ile618Asn/nonsenseN/N (Pancreatic sufficient)
Splice site/N N/N (bronchiectasis x2)p.Phe508del/N (bronchiectasis)
Overall pick-up rate 13/18 (72%)
Pick up rate 13/15 (87%) excluding bronchiectasis
?CF
Summary
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
90% 92.5% 98%
2 mutations 81% 85% 96%1 mutation 18% 14% 4%0 mutations 1% 1% <1%