How much CFTR do we need to double? the 2789+5G->A tipping point

  • How much CFTR do we need for a therapeutic effect?
    Doubling the Open probability: Will it be enough?
    (The 2789+5G->A tipping-point)

A little preface:
Splicing mutations cause the formation of an aberrant protein fraction and a correct wild type
cftr protein fraction. The amount of correctly spliced protein is variable among individuals with the
same mutations, and may vary from organ to organ; thus we can have different phenotypes even in
the same mutation subpopulation. Moreover, in this text we won’t speculate about the effect of any
potentiators on the aberrant protein produced. We will merely speculate on what would be the effect
of doubling the activity of wild type cftr protein that make it to the surface.
Based on the observations of Van Goor, Sloane Rowe and Yu ( as mentioned previously here ), CFTR (the only protein with channel activity among the family of ABC transporters) would double its open probability in the presence of a potentiator (Vx-770, Ivacaftor). This would result in an increase in the Cl- conductance activity of about a factor 1.5-2. This notion is confirmed by Jih and Hwang
from the University of Missouri-Columbia, who underline that “the open time of WT-CFTR (wild type CFTR) is significantly prolonged at high [ATP] (882 ± 141 ms, n = 25 in 10 mM versus 438 ± 34 ms, n = 17 in 100 μM) in the presence of VX-770” (Poster 205, "UNRAVELING THE MECHANISM OF VX-770", NACF, Orlando 2012).

Would this translate into a therapeutic effect?
The more normal protein is produced, the better the therapeutic outcome for the patient.[ about mortality or about lung and pancreatic function or about pathogens acquisiton)
Here is an interesting graph showing the qualitative correlation between % wild type CFTR (WT-CFTR) and disease outcomes [Diagnosing cystic fibrosis: blood, sweat, and tears. COLIN WALLIS]

Then, how much correct protein is produced in milder phenotypes?
Let’s focus on the splicing mutations that have some residual function. Given splicing variability, the amount of WT-CFTR can in theory vary between different mutations, patients or even organs. Therefore it is hard to correctly answer such question. However it is still possible to make some general estimates looking at a list of splicing mutations that surely produces some wild type protein on their surface [Castellani]

If one just takes into account the airway disease and ignores important influences such as: NMDecay, transcomplementation and an eventually occurring aberrant isoform activity (clearly an oversimplification), CFTR-WT quantity for a milder lung disease should be in a range from 4 to 10%. [ AOTC slides, page 117].

Some authors suggest that the threshold is >5% for a milder decline and >10% for no pulmonary involvements (read it as “cure”).

Data from Davis PB. Cystic fibrosis since 1938. Am J Respir Crit Care Med 2006;173:475–82

Potentiators can thus transform a severe phenotype into a mild phenotype (for those with little to no residual CFTR on the surface) and a mild-CF phenotype in a normal phenotype. (CBAVD and sinusitis are nothing compared to lung disease consequences of CF. This prompts many scientists prefer to define this as CFTR-related disease, rather than proper CF). This would consist in a prolonged lifespan with a slower progression of the disease or no CF at all.

Sousa M, Servidoni MF, Vinagre AM, Ramalho AS, Bonadia LC, et al. (2012) Measurements of CFTR-Mediated Cl− Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis. PLoS ONE 7(10): e47708. doi:10.1371/journal.pone.0047708

Note that in the graphic, 10-60% for the non classic CF has to be read as 10-30%

Assuming a two-fold increase in CFTR activity, we could imagine that heterozygous phenotype made up of 1 mild splicing mutation and 1 severe mutation (0% CFTR), would translate into a “two mild mutations” phenotype (or a mild mutation homozygous).
Many authors in the past described homozygous individuals with splicing residual mutations. These patients have been diagnosed in their adulthood, and their symptoms classified as very mild or absent [Noone, Pue, Zhou, et al.: The 5T Variant in Intron 8 Causes CF-type Lung Disease or The Novel CFTR Mutation A457P in a Male with a Delayed Diagnosis of Cystic Fibrosis Or Late diagnosis defines a unique population of long-term survivors of cystic fibrosis etc].

The 2789+5G->A Tipping point.

As represented in the figure above [Van goor], 2789+5G->A is the splicing mutation with less functional CFTR available and can represent the tipping point among splicing mutations for what potentiation result to expect [G551D, R117H and 2789+5G->A were the targets of the very first clinical trial testing Vx-770 on Cystic fibrosis patients ( ].
Interestingly, recently in Germany, two siblings with genotype DF508/2789+5G->A were reported to be as the most longliving patients among those with CF in the world. (Cystic Fibrosis in 65- and 67-Year-Old
Siblings Respiration 2006;73:698–704
This mutation allow 1 to 15% of Wild Type CFTR to be made, coupled with a 836X isoform aberrantly spliced [CFTR protein analysis of splice site mutation 2789+5 G-A Van Barnevald et alii ].
Highsmith et alii in 1997 [Identification of a Splice Site Mutation (2789 + 5G>A) Associated With Small Amounts of Normal CFTR mRNA and Mild Cystic Fibrosis HUMAN MUTATION 9:332.338 (1997)] quantified the correct fraction in 4%. In his paper, a consanguineous family is analyzed and among the 8 patients with a CF genotype (3 2789+5G->A/2789+5G->A and 5 DF508/2789+5G->A) collected mean FEV1 was 70.3 +/. 9 % at a mean age of 36.9 +/- 6.2 Years. Moreover, FEV1 data for the three homozygous individuals are 96%, 72% and 68% at 26, 62 and 52 years respectively. To note that these values were collected before 1997 meaning that no Pulmozyme ®, Hypertonic saline or advanced antibiotic treatments were available during lifespans of the observed cases.
What could be NOW, the effect on this and other CFTR mutation with residual function if potentiated?

Future perspectives
In this particular scientific era, such theoretical specualtions, paved the way for a genotypic-phenotypic approach in finding a therapy for a larger spectrum of CFTR mutations. This newer group of “treatable individuals” have a residual CFTR function on the cell surface, putting together those with a mutant CFTR-form with less activity (oversimplifying: Class IV), those with some wt-CFTR proteins on the cell surface but in less quantity ( oversimplifying: Class V) and those with the right quantity of a working CFTR on the cell surface, but with unstability defect and enhanced turnover (oversimplifying: Class VI).

[Reversing the Basic Defect: A Vision for the Future, Rowe and Skach, 2012]

A pilot trial-of-1 study with ivacaftor is actually ongoing on “residual” mutations. [ ] If this study will be successful, the theoretic range of activity of potentiators could be painted as in the following slides:

[Reversing the Basic Defect: A Vision for the Future, Rowe and Skach, 2012]

Further evidences: analyzed the resulting data of 2 clinical trials that included G551D patients. Those combined with splicing mutations resulted in a bigger clinical improvement. Click Here

This site has no commercial purposes and it's not involved, linked or sponsored by any Pharmaceutical industry.