WHAT DID WE GAIN FROM CLONING THE CF GENE?
Eitan Kerem
Department of Pediatrics and CF Center,
Hadassah University Hospital,
Jerusalem, Israel
Cystic fibrosis (CF) is caused by mutations in the CFTR gene which was cloned and
sequenced twenty years ago. Today we know that the protein encoded by the gene is a
chloride channel located in the apical membrane of exocrine epithelial cells. It has a
molecular weight of ~170 kDa and comprises 1480 amino acids which are divided into
five domains, i.e., two membrane-spanning domains (MSD1 and MSD2), each
composed of six trans-membrane segments (TM1 to TM12) that form the channel, two
nucleotide-binding domains (NBD1 and NBD2), capable of ATP hydrolysis, and a
regulatory domain (R), which contains numerous phosphorylation sites. The protein
structure indicates that CFTR belongs to the ATP-binding cassette transporter proteins.
Phosphorylation of sites in the R domain by protein kinase A, regulated by cyclic
adenosine monophosphate (cAMP), and the hydrolysis of ATP by NBDs, are essential
for activating the chloride channel. This knowledge has not only enhanced our
understanding of the mechanism of CF pathology, but has also provided explanations
for phenotypic variations. Based on the known sequence of the gene, genetic testing
has refined our ability to identify patients with CF and CF-related illnesses. Genetic
mutations are grouped into 6 classes (I-VI) that can be directly related to the quantity
of CFTR protein produced in the cells. The level of CFTR protein has a straight
implication on the severity of the disease and may explain organ-specific sensitivity to
the presence of normally functioning CFTR. Furthermore, seemingly organ-specific
manifestations of CF, such as congenital bilateral absence of the vas deferens
(CBVAD) can be accounted for by a mechanism based on the quantity of CFTR
protein in cells.
One of the earliest efforts, following the cloning of the gene, was invested in
identifying the spectrum of disease-causing mutations. As of today over 1600
mutations are listed in the CFTR mutation database which in most countries is used as
the basis for genetic testing. Increased usage of carrier screening and prenatal
diagnosis has lead to a decline in the incidence of CF. Newborn screening (NBS) has
been adopted in several countries leading to early diagnosis. Although the aim of NBS
is to improve the life expectancy of patients with CF, it is probably associated with a
decreased incidence in CF births as parents may opt not to have more children with
CF.
Many studies were already performed aiming to identify modifier genes for different
phenotypes of CF using two main approaches. The first is analysis of candidate genes
based on our current knowledge. By identifying modifiers of lung disease, already 40
candidate immune or inflammatory genes were studied for their effect on the severity
of lung disease. Two genes, transforming growth factor b1 (TGFB1) a cytokine and
mediator of fibrosis and mannose binding lectin 2 (MBL2), showed a possible effect
on lung disease or infection prevalence. In recent years significant progress in
technology and computational tools lead to techniques which enable association
studies on the entire human genome by using a large number of patients. Recently, a
new modifier gene for CF lung disease namely IFRD1was identified. This gene acts
in a histone-deacetylase (HDAC)-dependent manner to mediate transcription of other
genes and is expressed during terminal neutrophil differentiation.
The knowledge gained so far has led to new therapeutic approaches that address key
factors of cystic fibrosis pathophysiology. Past therapeutic successes were largely
based on targeting the consequences of the cystic fibrosis trans-membrane regulator
dysfunction, such as mucous retention, infection, and inflammation, but new therapies
may be able to address the underlying abnormality rather than its downstream effects.
The efficacy of these treatments still needs to be established, but early studies of
several compounds look promising.
In summary the cloning of the CFTR gene over 20 years ago provided us with tools
for diagnosis of atypical and equivocal presentations, carrier testing for prenatal
diagnosis and prevention of births of children with CF. This led to a decrease in the
number of new cases in most countries. Understanding the molecular function of
CFTR, and the mechanisms by which the different mutations cause CF led to the
development of new therapies which are currently in different stages of development
and in clinical trials.