Saturday, March 31, 2012

Calling All Heterozygotes!

I'm so happy to introduce my guest blogger, Sandy Castle del Conte BA, MS, who brought this research to my attention and produced the following explanation of something I didn't realize was possible... 

Reasons why Kalydeco may work for 'Class I and Class II' Cystic Fibrosis mutations
I read a comment made by someone on a social media website that went something like this: we don't know how Kalydeco could help someone with a Class I and a Class II Cystic Fibrosis mutation.  My thought was, well, some researchers have a pretty good idea of how it could happen, and I would like to share what I have learned about their research.

First and foremost, I have to caution that the research described here does not guarantee that Kalydeco will work for people carrying Class I and Class II mutations, nor anybody with a mutation different than G551D; I attempt to explain how CF-causing CFTR proteins could traffic to the cell surface, but even if they traffic to the surface, they still could be very unstable, and still may not be potentiated by Kalydeco. The research described below has only been conducted in laboratories, not in people. I do not intend to create high hopes and expectations, or guarantee results; I only want to provide a reasonable explanation of how it may happen. 

The data are pretty clear on what Kalydeco does; Vertex researchers have reported that in their laboratory studies, they have found that '[Kalydeco] has a similar effect on all [tested] CFTR forms with gating defects' (Yu et al., 2012). Also, they have found Kalydeco potentiates CFTR from Classes II through V in the laboratory, including F508del, as well as 'multiple unclassified CFTR mutant forms' (Van Goor et al., 2010). Kalydeco has been able to potentiate a multitude of mutated CFTR proteins that make it to the cell surface.

CFTR proteins currently categorized within Class I and II cystic fibrosis mutations are described as usually not trafficking from the endoplasmic reticulum (ER) of the cell to the cell surface. These categorizations, however, do not take into consideration the interaction between mutant CFTR proteins.

Many people with cystic fibrosis are heterozygous; two different CFTR proteins are co-expressed in their cells.

There are many laboratory studies demonstrating that when two different CFTR mutant proteins are co-expressed, mutant CFTR proteins move to the cell surface that would not traffic when expressed alone because of transcomplementation between the mutant proteins. 

Transcomplementation has been shown to occur in the laboratory, and researchers are continuing to study it as a form of gene therapy. I have found no scientific reports of transcomplementation occurring  naturally (in vivo), but it is possible that in at least some CF heterozygotes, transcomplementation is occurring, resulting in CFTR proteins at the cell surface.

CFTR mutants that have been demonstrated to be transcomplemented, resulting in maturation, and thus  trafficking of CFTR proteins, include: F508del (Cebotaru et al., Cormet-Boyaka et al., 2004, 2008, 2009, 2011, Owsianik et al, 2003, Sun et al., 2008), A455E (Cebotaru et al., 2009), and H1085R (Cormet-Boyaka et al., 2004). Even trafficking of wild-type (normal, non CF-causing) CFTR has been shown to increase due to transcomplementation with mutant proteins (Cebotaru et al., 2006, 2008).  These CFTR proteins have been transcomplemented by a number of different proteins: wild-type and mutant CFTR proteins truncated (cut/missing sections) at the beginning, middle, or end, as well as truncated and full length proteins with amino acid deletions or substitutions.

The way that CFTR proteins interact and transcomplement is not entirely understood and seems to differ among CFTR mutations.  It seems that some CFTR mutants are targeted by and occupy the ER quality control system, allowing the second CFTR protein to escape to the cell surface (Sun et al., 2008), while other mutants actually associate with each other, 'most likely via a bimolecular interaction' (Cebotaru et al., 2011), altering and creating a protein that can traffic to the surface.

There is more to the story, but we'll save the rest for another day.

Many thanks go to Rebecca, for collaborating with me to 'translate' the following information, and for agreeing to post this on her blog.

Much appreciation also goes to Dr. Kevin Kirk, Professor and Vice Chair for the Department of Cell, Developmental and Integrative Biology, at University of Alabama at Birmingham, for thoroughly and clearly answering the many questions I have had for him, promptly answering my emails, conducting and supporting research on cystic fibrosis, and for giving me the citation to the first transcomplementation paper I ever read.

Sandy Castle del Conte, BA, MS

Side Notes: 

If you are interested in looking at mutations in detail, the Cystic Fibrosis Mutation Database has information on the currently known CFTR mutations ( Please note that the numbering of nucleotides and amino acids indicated in the papers cited below is slightly different from that found on the Cystic Fibrosis Mutation Database. The c.DNA name is the most accurate way to find the location of mutations on this website.

A tidbit of data I found interesting is: CFTR under 'constant stimulation' is degraded faster (Lewarchik et al., 2008).

Rebecca's Take on Transcomplementation
Is your head spinning yet?  I realize this is very technical information, but an extremely interesting line of research.  Especially since we now have an FDA approved drug like Kalydeco, which has been shown in the lab to have the ability to potentiate (open the gate) for any CFTR that succeeds at arriving to the cell surface.  Getting that protein to the cell surface is now an important goal in research and the reason why drugs like VX-809 and VX-661 are being studied right now.  The concept of “transcomplementation” represents an alternate process by which some CFTR might successfully reach the cell surface for some CF heterozygotes (2 different mutations). 

To shed a little light on this complicated process, an analogy is in order.  In a previous blog, I referred to CFTR as a piece of genetic origami.  For the purposes of this explanation, let’s envision the CFTR protein like a jigsaw puzzle.  There is a factory inside epithelial cells that produces jigsaw puzzles (CFTR proteins).  The jigsaw puzzles are churned out and sent to quality control (the endoplasmic reticulum).  Quality control ensures that all the pieces are present and fit together properly before each puzzle is shipped out to the customer (the cell surface).    In healthy individuals, this process happens smoothly.  The puzzles have all their pieces, pass quality control, and are shipped out to the customer.  In individuals with cystic fibrosis, some of the pieces of the puzzle are missing.  Each mutation of cystic fibrosis causes a different piece of the puzzle to be missing.  Let’s say that, for example, all delta F508 mutations are missing the upper right hand corner of the puzzle.  All delta 264 are missing the middle piece, etc…  Transcomplementation refers to an interaction that can occur when two different mutations (puzzles) exist in the cell (heterozygous).  In other words, if a puzzle missing the upper right hand corner gets together with a puzzle that HAS the upper right hand corner but is missing the middle piece…they might share pieces until they come up with a single complete puzzle that could successfully pass through quality control and get shipped.  It is essential that the missing piece from one puzzle be present in the other puzzle (mutation) for transcomplementation to occur.  This explains why this phenomenon doesn’t happen with homozygous mutations.  If the mutations are the same, all the puzzles in the cell would be missing the same piece--so sharing wouldn’t get you any closer to a complete puzzle!        

Another theory is that when two different CF mutations are present, it is possible for quality control to become somewhat distracted while counting and sorting all the pieces of the first screwed up puzzle.  While quality control has it’s back turned, dealing with the first mutation, some of the second mutation can potentially sneak through the endoplasmic reticulum without even being checked for quality, and successfully reach the cell membrane. 

Again, the idea is that once CFTR reaches the cell membrane, we now have a drug, Kalydeco, which can “turn on” the action of that protein.  I’ve read of a few cases of off-label use of Kalydeco in mutations outside of Class III (gating), that have described improvement, when according to our current classification, they shouldn’t have any available CFTR to potentiate (  The idea I want to put out there is that, while some CFTR mutations aren’t technically SUPPOSED to produce any protein on the cell surface, sometimes there may actually be a small amount because of processes like transcomplementation.  The more I learn about the genetics of cystic fibrosis, the more I realize that this disease is as individual to each patient as their fingerprint.  It may be very difficult to predict what is happening within the cells of a CF patient, because some people spontaneously have interactions like “transcomplementation” occurring, that are new concepts to the world of CF science.  I was very excited when Sandy approached me with this research.

The therapeutic implications of transcomplementation are that fragments of genes with the correct missing puzzle pieces might be able to be inserted into the mix, to allow a person’s body to produce complete proteins that fully “mature” and reach the cell surface.  Also, there is the potential that the pool of patients that could actually receive some benefit from Kalydeco, might be much larger than originally anticipated.  Again, this is certainly NO GUARANTEE that Kalydeco would work for you just because you are heterozygous with a Class I or II mutation…but it certainly brings to light the idea that it is possible under certain circumstances.  That is what this blog is all about!  Possibilities!  I want to thank Sandy for this wonderful piece of work and dozens of useful references(bottom of page).  I look forward to working with her again in the future as we delve even deeper into this topic.

Lastly, my blog wouldn’t be complete without a quick update on Brady.  He is doing fantastic!  Sinuses are still clear, he seems to be feeling amazing, full of energy, and gaining weight.  I haven’t been giving updates as frequently, but only because there is nothing new to report!  In the CF world, nothing new= good news!  He honestly seems like a new kid since beginning Kalydeco.   We are looking forward to the Chest CT scan in a few weeks and will give a full report on the results!
Carroll, T. P., Marcelo M. Morales, Stephanie B. Fulmer, Sandra S. Allen, Terence R. Flotte, Garry R. Cutting and William B. Guggino. Alternate Translation Initiation Codons Can Create Functional Forms of Cystic Fibrosis Transmembrane Conductance Regulator. J Biol Chem. 1995 May 19;270(20):11941-6.
Cebotaru, L., Terence R. Flotte and William B. Guggino. AAV [Delta]264CFTR Enhances Maturation of [Delta]F508CFTR and wt CFTR Expression. Molecular Therapy (2006) 13, S193.

Cebotaru L, Vij N, Ciobanu I, Wright J, Flotte T, Guggino WB. Cystic fibrosis transmembrane regulator missing the first four transmembrane segments increases wild type and DeltaF508 processing. J Biol Chem. 2008 Aug 8;283(32):21926-33.
Cebotaru, L., and William Guggino. Rescue of A455E CFTR by temperature, small molecule correctors and transcomplementation. Journal of Cystic Fibrosis. June 2009, 8, Supplement 2, pg. S17-S17.

Cebotaru, L.; Woodward, O.; Guggino, W.B. A truncation mutant of CFTR, 27-264-CFTR, rescues  both trafficking and chloride channel function of F508 CFTR by transcomplementation. Pediatric Pulmonology. October 2011. Volume 46, Issue S34, Page 214.

Cormet-Boyaka, E., Michael Jablonsky, Anjaparavanda P. Naren, Patricia L. Jackson, Donald D. Muccio, and Kevin L. Kirk. Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation. Proc Natl Acad Sci U S A. 2004 May 25; 101(21): 8221–8226.

Cormet-Boyaka, E., Jeong S. Hong, Bakhram K. Berdiev, James A. Fortenberry, Jessica Rennolds, J. P. Clancy, Dale J. Benos, Prosper N. Boyaka, and Eric J. Sorscher. A truncated CFTR protein rescues endogenous ΔF508-CFTR and corrects chloride transport in mice. The FASEB Journal. 2009 November; 23(11): 3743–3751.
Fischer AC, Smith CI, Cebotaru L, Zhang X, Askin FB, Wright J, Guggino SE, Adams RJ, Flotte T, Guggino WB. Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates. Mol Ther. 2007 Apr;15(4):756-63.
Flume, P.A.; Borowitz, D.; Liou, T.; Li, H.; Yen, K.; Ordoñez, C.; Geller, D.E.5VX-770 in Subjects with CF and Homozygous for the F508DEL-CFTR Mutation. Pediatric Pulmonology.  October 2011. Volume 46, Issue S34. Pp 284 – 285.
Flume, Patrick A., MD (, Theodore G. Liou, MD (, Drucy S. Borowitz, MD (, Haihong Li (, Karl Yen, MD (, Claudia L. Ordoñez, MD (, David E. Geller, MD ( and for the VX08-770-104 Study Group. Ivacaftor in Subjects with Cystic Fibrosis who are Homozygous for the F508del-CFTR Mutation. Chest. March 2012. [Epub ahead of print].
Lewarchik, C. M., Kathryn W. Peters, Juanjuan Qi, and Raymond A. Frizzell. Regulation of CFTR Trafficking by Its R Domain. J Biol Chem. 2008 October 17; 283(42): 28401–28412.
Owsianik G, Cao L, Nilius B. Rescue of functional DeltaF508-CFTR channels by co-expression with truncated CFTR constructs in COS-1 cells. FEBS Lett. 2003 Nov 6;554(1-2):173-8.
Ramalho AS, Lewandowska MA, Farinha CM, Mendes F, Gonçalves J, Barreto C, Harris A, Amaral MD. Deletion of CFTR translation start site reveals functional isoforms of the protein in CF patients. Cell Physiol Biochem. 2009;24(5-6):335-46.
Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD. Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17891-6.
Sun, F., Zhibao Mi, Steven B. Condliffe, Carol A. Bertrand, Xiaoyan Gong, Xiaoli Lu, Ruilin Zhang, Joseph D. Latoche, Joseph M. Pilewski, Paul D. Robbins and Raymond A. Frizzell. Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia. The FASEB Journal. 2008;22:3255-3263.
Van Goor, F.; Yu, H.; Burton, B. The investigational CFTR potentiator, VX-770, potentiated multiple CFTR forms in vitro. Journal of Cystic Fibrosis. 2010. Volume 9, S14.
Yu, H., Bill Burton, Chien-Jung Huang, Jennings Worley, Dong Cao, James P. Johnson Jr., Art Urrutia, John Joubran, Sheila Seepersaud, Katherine Sussky, Beth J. Hoffman, Fredrick Van Goor. Ivacaftor potentiation of multiple CFTR channels with gating mutations. Journal of Cystic Fibrosis. Available online 30 January 2012.


  1. I stumbled onto this blog seeing the defect A455E mentioned. A lot of really good info!
    It's really too bad the upcoming trials are so limited with Kalydeco to just gating defects (outside those combined with VX-809). The evidence out there implies it could be helpful with any defect that gets the protein to the cell surface (
    With the positive mid-term VX-809 results hopefully leading to the continuation of a phase III trial and eventual FDA approval, I hope that Kalydeco can become an off-label prescription someday for those with defects so rare they will never be part of a clinical trial.