1. Genes Genomes and Personalized Medicine
CFTR … and Kalydeco
Cystic Fibrosis Transmembrane Conductance Regulator
Small molecule drug…
…developed by Vertex…
…with support from the Cystic Fibrosis Foundation…
…that works for CF patients who have the G551D mutation.

2. CF is the most common lethal genetic disease in Caucasians.
CF is the most common lethal genetic disease in Caucasians.

4. The CFTR Gene was first cloned and sequenced in 1989…
The CFTR Gene was first cloned and sequenced in 1989…. …… by Lap-Chee Tsui and Francis Collins
Riordan, J.R. et.al., Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA. Science, 245, pp (1989)

5. Nucleotide Binding Domain Nucleotide Binding Domain
CFTR is a member of ATP-binding cassette (ABC) transporter superfamily.
Outside of cell
MEMBRANE
Six Transmembrane
Alpha Helices
Six Transmembrane
Alpha Helices
Cytoplasm
Nucleotide Binding Domain
Nucleotide Binding Domain

6. The first six transmembrane helices (TM1, TM2, TM3, TM4, TM5, TM6) of CTFR
If each TM sequence (21 aa) is an alpha helix – and there are 3.6 amino acids per turn of the helix….each TM helix would be ~6 turns long. Since each turn of the helix is 5.4A long….6 turns is 32 A or 3.2 nm. This is just long enough to span the thickness of a lipid bilayer

7. The CFTR protein consists of the following domains….
CFTR mRNA map N C
TM 1-6
NBD1
TM 7-12
NBD2
Regulatory Domain

8. 2 important mutations map to the NBD of CFTR
G551D is here
The F508del is here

9. OMG! …. the F508del mutation of CFTR
Understanding the F508del mutation of CFTR is a mind-blowing experience! Hang on……
----CAUCUUUGGUG----
I F G
----CAUUGGUG----
I G
507
509
508
3 base deletion
The nucleotide sequence of the normal gene in this region is:
While you might assume that the F508 mutation is UUU – in fact it is CUU.
The CUU deletion changes the sequence to
To put this in words,…. The 3rd position of codon 508 becomes the 3rd position of codon And since both AUC and AUU both encode Isoleucine (I) – this 3base deletion results in a clean deletion of F508, with no other change in amino9 acid sequence. Is this a great country or what?

10. Potential PKA (Protein Kinase A) target sites in the R-domain of CFTR:
The Regulatory Domain of CFTR contains many “dibasic” target sites that are phosphorylated by Protein Kinase A (PKA) and Protein Kinase C (PKC). Shown below --- a listing of the dibasic sites – and flanking sequence – found in this region of CFTR.
As you “look for patterns” (a Crosscutting Concept in the NGSS), can you see a larger consensus sequence that defines this phosphorylation site?

11. Sanger Sequencing Gel – of the region surrounding the F508 codon
Normal CF
G A T C
Riordan, J.R. et.al., Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA. Science, 245, pp (1989)

14. CFTR Model,… 1989
Riordan, J.R. et.al., Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA. Science, 245, pp (1989)

15. CFTR Model,… 2000
Akabas, M.H., Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem. 275, pp (2000)

16. CFTR Model,… 2000
Akabas, M.H., Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem. 275, pp (2000)

17. CFTR Model,… 2006
Gasby, D. et.al., The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440, pp (2006)

18. CFTR Model,… 2010
Atwell et.al., Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant. Prot. Eng. Design & Selection 23, pp (2010)