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%