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The C linical and F unctional TR anslation of CFTR (CFTR2) Project

The C linical and F unctional TR anslation 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. R1077P. N1303K. D1152H. R117H-5T/7T. S1251N.

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The C linical and F unctional TR anslation of CFTR (CFTR2) Project

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  1. The Clinicaland Functional TRanslation of CFTR(CFTR2) Project Garry Cutting on behalf of the CFTR2 project team

  2. CF Transmembrane conductance Regulator (CFTR) Serohijos A. W. R. et.al. PNAS;2008;105:3256-3261

  3. CFTRdele 22,23 R1077P N1303K D1152H R117H-5T/7T S1251N 3905insT 711+5G>A G542X DF508 F1074L p.Phe508del E60X R349L M470V Y569D R668C Q220X G551D V520F P67L P205S 3849+10kbC>T

  4. The genetic testing gap Fraction of allmutations that occur in patients with CF Fraction of allmutations reported in the CFTR gene Fraction of CF patients with bothmutations identified 70% 49% <0.1% F508del 1.2% 85% 72% 23 ACMG mutations

  5. 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

  6. A new repository for clinical data associated with CFTR mutations CFTR1 (CF Mutation Database) Gene information 1893 mutations Link by mutation CFTR2 Clinical information 39,545 patients

  7. Contributors to CFTR2

  8. Summary of clinical data collected CFTR2 Database 39,545 patients 23 registries/clinics Pancreatic Status Lung Function (FEV1%predicted) Sweat Chloride Concentration CFTR Genotype 16,204 patients missing PFT data 14,403 patients missing sweat data 5276 patients with 1 mutation unknown 9309 unknown 1674 patients with both mutations unknown 250 measurements excluded 3 measurements <5 % predicted excluded 70,466 CF chromosomes with a mutation identified 23,338 patients 24,892 patients 30,236 patients

  9. 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

  10. How do we determine which mutations cause CF and which ones don’t?

  11. 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

  12. Clinically consistent mutation • Elevated sweat chloride concentration • Reduced FEV1 % predicted • Exocrine pancreatic disease • Infection with Pseudomonas aeruginosa • Other features (meconiumileus, male infertility (CBAVD)

  13. Sweat chloride concentrations in 10,108 F508delhomozygotes Mean 103 + 16.8 mEq/L 60 mEq/L Number of patients Sweat chloride concentration

  14. How do we isolate the effect of a mutation in patients that carry two mutations? 7 7 CFTR

  15. How do we determine which mutations cause CF and which ones don’t? Clinically consistent mutation Functionally consistent mutation

  16. Predicted effect of 160 mutations upon CFTR function Change in one amino acid

  17. CFTR Function Expert Committee Margarida Amaral, PhD - University of Lisbon, Portugal Bob Bridges, PhD - Rosalind Franklin University, Illinois, US GergelyLukacs, MD - McGill University, Montreal, Canada David Sheppard, PhD – Bristol University, UK Phil Thomas, PhD - UT Southwestern, Dallas, US

  18. 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 expression FRT stable expression CFTR splicing (Margarida Amaral) CFTR minigene plasmids HEK293 stable expression CFBE41o- stable expression (planned) In vivo (when possible) mRNA level: Quantitative PCR Site-directed mutagenesis Cell line generation CFTR Maturation: Western Blot CFTR Function: Ussing Chamber FRT cell lines created analyzed for 57 missense and 2 deletion mutations

  19. How do we determine which mutations cause CF and which ones don’t? Clinically consistent mutation Functionally consistent mutation Genetically consistent mutation

  20. 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

  21. 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

  22. Improving genetic testing for CF Fraction of allmutations that occur in patients with CF Fraction of allmutations reported in the CFTR gene Fraction of CF patients with bothmutations identified 1.2% 85% 72% 23 ACMG mutations 8.4% 97% 90% 160 CFTR2 mutations

  23. What is the best way to present this information in a public database?

  24. 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

  25. G551D I148T D1152H

  26. 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

  27. CFTR2 Team Julian Zielenski Vertex Pharmaceuticals and NIDDK R37 DK44003

  28. Thanks to the CF clinical and research community for making this project possible

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