DNA analysis Molecular genetic testing for cystic fibrosis

1
DNA analysisMolecular genetic testing for cystic
fibrosis
Carolyn TysoePrincipal Clinical
ScientistRoyal Devon Exeter NHS Foundation
Trust
2
Outline

• DNA basics structure, function, types of
  mutation
• Mutation detection
• Introduction to cystic fibrosis and CFTR gene
• CFTR mutations
• Testing strategy
• Case study

3
DNA transcription and translation
5
3
DNA
G
3
5
mRNA
nucleus
cytosol
4
Effects of single base substitutions
Wild type
Glu
Glu
Lys
Cys
Phe
Cys
Lys
Cys
Phe
Cys
Met
ATG
AAG
TGC
TTC
TGC
GAG
Stop
5
Splice site mutations
GT
GT
GT
AG
AG
AG
DNA
DNA
Normal spliced mRNA
DNA
Exon skipping
DNA
Intron inclusion
DNA
Use of a cryptic splice site
6
Deletions and insertions
of one or a few base pairs
Frameshift
Arg
Glu
Lys
Cys
Phe
Cys
Met
Lys
Cys
Ser
Ala
ATG
AAG
TGT
TCT
GCG
AGG
7
Search for unknown mutations eg sequencing
Look for known mutations eg OLA
Mutation detection methods
Look for single or multi-exon deletions eg MLPA
8
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

9
Method depends on mutation spectrum of gene
CFTR gene and cystic fibrosis
10
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?

11
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

12
Cystic Fibrosis

• What are the major clinical features?
• Any additional features?

13
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

14
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

15
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)

16
CFTR gene to protein
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CFTR mutations

• What is the mutation spectrum?
• What is the most common mutation?

18
CFTR mutations
42 10 16 2 13 0.5 15
1546 mutations listed on mutation
database www.genet.sickkids.on.ca/cftr
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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
20
Most common CFTR mutation

• p.Phe508del (?F508)
• 3bp deletion (CTT)
• Deletes phenylalanine at codon 508
• 75 in UK population
• 66 in world population

21
Mutation classes
Increasing severity in phenotype
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Variable splicing of exon 9
5T or 7T or 9T
GTGTG(T)AACAG
DNA
Exon 8
Exon10
Exon 9
Intron 8
Intron 9
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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
24
?F508/R117H genotypes
?F508/R117H (7T) CBAVD or
Pancreatic sufficient CF
25
Spectrum of CFTR disease

• Estivill et al Nature Genetics 1996

26
CF testing at Exeter

• What referral reasons do we see?
• What molecular tests do we offer?

27
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

28
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
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Multiplex of 15 PCR reactions
1
9
10
2 3
11 12
13 14a
19
20
21
18
22 23 24
4
7 8
16 17a 17b
14b 15
5 6a 6b
Mutant
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Normal result
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Heterozygous p.Phe508del
01. Patient1 Blue
I507
V520
1535
1024
F508
01. Patient1 Green
803
1500
1000
500
01. Patient1 Yellow
32
Homozygous p.Phe508del
01. Patient1 Blue
I507
V520
1535
1024
F508
01. Patient1 Green
1623
1500
1000
500
01. Patient1 Yellow
33
Our inheritance, our futureRealising the
potential of genetics in the NHS
Genetics White Paper 2003

• The NHS should lead the world in taking maximum
  advantage of the application of the new genetic
  knowledge for the benefit of all patients
  

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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

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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

36
Testing strategy extended CFTR analysis for
SCOBEC network
6 million to achieve

Salisbury
Cambridge
Oxford
Cardiff
Bristol
Exeter
37
Modernisation of Exeter Lab
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Reporting time data

• 3 days for urgent samples
• 10 days for known mutation
• 40 days for unknown mutations

39

• Testing strategy extended CFTR analysis for
  SCOBEC network

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Extended CFTR testing

1. Sequencing of entire gene (27 exons)

2. Dosage analysis
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DNA Polymerase
(a)

5 C C T G C A G G C T G G G C G 3 G G A C G T
C C G A C C C G C C T G T G C 5
C C T G C A G G C T C C T G C A G G C T G C C T G
C A G G C T G G C C T G C A G G C T G G G C C T G
C A G G C T G G G C C C T G C A G G C T G G G C
G C C T G C A G G C T G G G C G G C C T G C A G
G C T G G G C G G A C C T G C A G G C T G G G C G
G A C C C T G C A G G C T G G G C G G A C A C C T
G C A G G C T G G G C G G A C A C C C T G C A G
G C T G G G C G G A C A C G
(b)
(c)
42
Partial or whole gene deletions are not detected
by sequencing

• When you sequence an exon how do you know how
  many copies there are?

• Need a quantitative (dosage) test

43
CFTR deletions and duplications

• 44 reported out of 1546 CFTR mutations (2.9) (CF
  mutation database)

Deletion
Duplication
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MLPA probes
PCR primer sequence Y
24 bp sequence specific probes
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Annealing of probes
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Ligation of probes
PCR primer sequence Y
A ligase enzyme ligates the 2 probes together
Only annealed probes will be ligated
47
Samples are heated to denature the probe from the
DNA
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Probe amplification
The probe is amplified using the common primer
pair
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All the probes can be amplified using the same
primer pair and PCR conditions
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MLPA Results - Electrophoresis
CFTR Duplication Exons 6b-10
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MLPA Results Spreadsheet analysis
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MLPA Results graphical display
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Testing for CFTR mutations
1995 1996 1997 2004
2006
CF4 CF12 CF31 CF33
CFTR entire gene 80 85 92.5
92.5 98 mutations
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Patient KF

• 4 year old boy
• Chest infections
• Abnormal liver ultrasound
• ?Cystic fibrosis

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Patient KF

• Routine test for CF Genotype

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KF is heterozygous for p.Phe508del
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Heterozygous p.Phe508del mutation in KF
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Patient KF

• Request for extended CFTR testing

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• Referral criteria for extended testing

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Guidelines for extended CFTR testing (Sequencing
and MLPA 700)
a)
Obligate Carrier (Clinical diagnosis of CF in
offspring)
Risk factors AND AND
1 phenotypic symptom (recurrent chest infections, pancreatic insufficiency or CBAVD) And/Or ve NBS (two high IRTs) 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.
b)
If unable to obtain sufficient sweat or NPD
unavailable, proceed if 2 phenotypic symptoms
or 2 of the and/or risk factors
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Patient KF

• Request for extended CFTR testing

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MLPA Result for KF
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Sequencing identified a heterozygous p.Glu585X
mutation in KF
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Heterozygous p.Glu585X mutation in KF
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Results to date
Clinical diagnosis CF
Obligate carrier
?CF
p.Phe508del/splice site (2) c.17661GgtA/splice
site p.Phe508del/truncating (3) p.Phe508del/misse
nse (3) c.3067_3072del6 p.Ile148Thr/missense c.3
71710kbCgtT/missense p.Arg1070Gln/N p.Ile618Asn/n
onsense N/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
66
Summary
90 92.5 98 2
mutations 81 85 96 1 mutation 18 14 4
0 mutations 1 1 lt1

• 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