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The Clinical and Functional TRanslation of CFTR
(CFTR2) Project
Garry Cutting on behalf of the CFTR2 project team
CF Transmembrane conductance Regulator
(CFTR)
Serohijos A. W. R. et.al. PNAS;2008;105:3256-3261
CFTRdele 22,23
N1303KD1152HR1077P
R117H-5T/7T
S1251N 3905insT
711+5G>A
G542X
DF508
F1074L
E60Xp.Phe508del
R349L M470V
Y569DR668C
G551D
Q220X
V520F
P67L
P205S 3849+10kbC>T
The genetic testing gap
Fraction of all
mutations reported
in the CFTR gene
<0.1%
Fraction of all
mutations that occur
in patients with CF
70%
Fraction of CF
patients with both
mutations identified
49%
F508del
1.2%
23 ACMG
mutations
85%
72%
Existing resources for CFTR mutations
The Toronto CF Mutation Database
• Mutation-driven: Information deposited by
genetic laboratories, primarily research
Online Mendelian Inheritance in Man (OMIM)
• Publication-driven: Information from manuscripts
authored by researchers
A new repository for clinical data
associated with CFTR mutations
Gene information
CFTR1
(CF Mutation Database)
1893 mutations
Link by mutation
Clinical information
CFTR2
39,545 patients
Contributors to CFTR2
Summary of clinical data collected
CFTR2 Database
39,545 patients
23 registries/clinics
CFTR Genotype
Sweat Chloride
Concentration
5276 patients with
1 mutation unknown
1674 patients with
both mutations
unknown
70,466 CF
chromosomes
with a mutation
identified
Lung Function
(FEV1%predicted)
14,403 patients
missing sweat
data
250 measurements
excluded
24,892
patients
16,204
patients
missing PFT
data
Pancreatic
Status
9309
unknown
3 measurements
<5 % predicted
excluded
23,338
patients
30,236
patients
Where did we start?
160 mutations are seen in 9 or more patients in
the CFTR2 database
• Allele frequency of 0.0001 or .01%
• This represents 97% of total identified CFTR
mutations
How do we determine which mutations
cause CF and which ones don’t?
Clinical Expert Committee
• Christiane De Boeck, MD, PhD - University Hospital of Leuven,
Belgium
• Peter Durie, MD - Hospital for Sick Children, Toronto, Canada
• Stuart Elborn, MD - Queen's University, Belfast, UK
• Phil Farrell, MD, PhD – Univ. Wisconsin, USA
• Michael Knowles, MD - University of North Carolina, Chapel
Hill, USA
• Isabelle Sermet, MD, PhD- Necker Hospital, Paris, France
Clinically consistent mutation
•
•
•
•
Elevated sweat chloride concentration
Reduced FEV1 % predicted
Exocrine pancreatic disease
Infection with Pseudomonas
aeruginosa
• Other features (meconium ileus, male
infertility (CBAVD)
2000
Sweat chloride concentrations in
10,108 F508del homozygotes
500
of patients
Number
60 mEq/L
0
1000
Frequency
1500
Mean 103 + 16.8 mEq/L
0
50
100
150
Sweat chloride
Sweat chloride concentration
200
250
How do we isolate the effect of a mutation
in patients that carry two mutations?
7
7
CFTR
How do we determine which mutations
cause CF and which ones don’t?
Clinically consistent mutation
Functionally consistent mutation
Predicted effect of 160 mutations
upon CFTR function
Change in
one amino
acid
CFTR Function Expert Committee
Margarida Amaral, PhD - University of Lisbon, Portugal
Bob Bridges, PhD - Rosalind Franklin University, Illinois, US
Gergely Lukacs, MD - McGill University, Montreal, Canada
David Sheppard, PhD – Bristol University, UK
Phil Thomas, PhD - UT Southwestern, Dallas, US
Functionally consistent mutation
CFTR procession and function (Fred Van Goor)
Fisher Rat Thyroid (FRT) cells expressing CFTR from single cDNA integration
Characterize the processing and function of CFTR
CFTR processing (Phil Thomas)
HeLa transient expressionCell line
Site-directed
mutagenesis
FRT stable expression generation
CFTR splicing (Margarida Amaral)
mRNA level:
Quantitative PCR
CFTR Maturation:
Western Blot
CFTR Function:
Ussing Chamber
CFTR minigene plasmids
HEK293 stable expression
CFBE41o- stable expression (planned)
FRT(when
cell lines
created analyzed for 57 missense and 2 deletion mutations
In vivo
possible)
How do we determine which mutations
cause CF and which ones don’t?
Clinically consistent mutation
Functionally consistent mutation
Genetically consistent mutation
Genetically consistent mutation
Fertile fathers of CF patients should
carry only one mutation that causes CF
Confirm that none of the clinically
and functionally consistent mutations
occur as the second mutation in a
father of a CF patient
Mutations occurring in at least 9 patients have a
frequency ~0.0012 (9/8400 genes without ACMG
mutations)
2000 ‘healthy’ CFTR genes in 2000 fathers provides
80% power to detect variants at 0.002 at type I error
rate of 0.05
How do we determine which mutations
cause CF and which ones don’t?
Clinically consistent mutation
Functionally consistent mutation
Genetically consistent mutation
CF-causing mutation
Improving genetic testing for CF
Fraction of all
mutations reported
in the CFTR gene
1.2%
Fraction of all
mutations that occur
in patients with CF
85%
Fraction of CF
patients with both
mutations identified
72%
23 ACMG
mutations
8.4%
160 CFTR2
mutations
97%
90%
What is the best way to present this
information in a public database?
CFTR2 Patient Advocacy Committee
Barbara Karczeski MS(Genetic Counselor)- Johns Hopkins DNA
Diagnostic Lab, Baltimore, MD
Michelle Huckaby Lewis, MD, JD (Ethics expert) – Berman Institute
of Bioethics/Genetics and Public Policy Center, Johns
Hopkins, Baltimore MD
Bruce Marshall, MD (CFF representative) - CF Foundation,
Bethesda, MD, USA
Juliet Page (Patient representative) - Annapolis, MD, USA
G551D
I148T
D1152H
Summary
• Data from nearly 40,000 CF patients into the
CFTR2 database have been instrumental in:
– Increasing the list of clinically, functionally and
genetically vetted ‘CF-causing’ mutations from 23 to
~160 (more to follow..)
– Providing complete CFTR mutation information on 9
out of 10 patients with CF
– Creating the infrastructure for new studies into the
relationship between CFTR function and the CF
phenotype
CFTR2 Team
Julian Zielenski
Vertex Pharmaceuticals and NIDDK R37 DK44003
Thanks to the CF clinical and
research community for making
this project possible