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Cystic Fibrosis and CFTR Gene
*
I- Background
II- Incidence
III-Clinical Manifestations
IV- Diagnosis
V- CFTR gene and its mutations
V-1. Introduction
V-2. The DF508 mutation
V-3. Spectrum of CFTR gene mutations
V-4. Genotype-phenotype correlations
VI- CFTR protein and its functions
VI-1. Structure of CFTR protein
VI-2. Function of Cl- channel
VI-2.1. Function of Cl- channel
VI-2.2. The CFTR protein, a multi-functional protein
VI-3. Correlations of mutations of CFTR gene with
the function of Cl-
channel
VI-3.1. Class 1: mutations altering the production
of the protein.
VI-3.2. Class 2: mutations disturbing the process of cellular maturation
cellular of the protein.
VI-3.3. Class 3: mutations disturbing the regulation of Cl- channel.
VI-3.4. Class 4: mutations altering the conduction of l Cl- channel.
VI-3. 4. Class 5: mutations altering the stability
of mRNA,
VI-3. 4. Class 6: mutations altering the stability of mature protein.
VII- References |
French
pdf version |
*
I- Background
- The early description of cystic fibrosis (CF) dates back to late 30s.
In 1936, Fanconi identified the association between the ìcongenital CF of
the pancreas and broncheactasis" shortly followed by Andersen who in
1938 gave the complete anatomo-pathologic description of CF.
- In 1953, Di Sant'Agnese described an excess of sodium chloride in the
sweat of children affected by CF. This discovery shortly leads to the use
of sweat chloride test, the only reliable diagnostic test of the disease
available to this date.
- In early eighties, the abnormality of transport of salts was precisely
described by Quinton (Quinton 1983) who explained the defect of permeability
of chloride ions (Cl-) in the affected epithelial cells of sweat glands,
and later by Knowles (Knowles 1983) who observed the same phenomenon in
the respiratory epithelium.
- In 1989, the CFTR gene, implicated in the CF, was isolated. This gene
is localized on 7q31 and contains 27 exons. The protein is composed of 1480
amino acids.
II- Incidence
The CF is the most common lethal autosomal recessive hereditary
disorder, worst in the European origin populations, affecting an average one
out of 2500 live births (= q2), i.e., one out of 25 (= 2pq) individuals is a
carrier of this disease. However, this frequency varies according to geographic
and ethnic origin of the patients.
III- Clinical manifestation
- The clinical presentation of CF varies among individuals from different
families as well as between individuals belonging to the same family. In
majority of cases the disease is diagnosed before adolescence, but some
remain asymptomatic till the adult age. Clinically and biologically, it
is not possible to distinguish the heterozygotes from the individuals not
carrying CF mutation.
- The clinical presentation varies with age. In 10% of the affected newborns,
it presents as meconium ileus (intestinal obstruction due to abnormally
thick meconium). Later the symptomatology involves two major organ systems,
respiratory and digestive, manifesting as repetitive respiratory infections
and signs of malabsorption.
- The respiratory involvement predominates. It is related to an obstruction
of bronchioles by thick and viscous mucous favoring the growth of microorganisms.
This explains the repeated respiratory infections by the opportunistic germs.
- The digestive involvement with exocrine pancreatic insufficiency (defect
in the production of enzymes) is seen in 85% of the patients, leading to
the obstruction of pancreatic ducts and thus lipoprotein malabsorption.
The state of exocrine pancreas, PI (pancreatic insufficiency) or PS (pancreatic
sufficiency), serves as a marker of the gravity of the phenotype, being
serious in the former than the latter. (Kerem 1990).
- The abnormality in the sweat glands leads to an excess of sodium chloride
secreted in the sweat, this loss of salt is usually responsible for acute
dehydration in case of exposure to heat.
- This disease involves some other organs particularly the reproductive
system and the liver.
- Atrophy and absence of vas deferens caused by obstructive azoospermia
renders 98% of men sterile, on the other hand 80% of affected females are
fertile.
- The hepatic involvement in 30% of cases starts as a hepatomegaly and
in 9% of cases as a hepatic insufficiency. It is due to the obstruction
of the intrahepatic or extrahepatic biliray tracts by compression at the
level of pancreas. In 2-5% of cases this further leads to biliary cirrhosis.
- Prognosis: If not treated, the median survival rate is 3 to 5 years.
- There is no effective treatment of CF, but the timely diagnosis and symptomatic
treatment has made it possible to increase the mean survival to 25 to 30
years.
- The symptomatic treatment is available in the from of respiratory physiotherapy,
antibiotic therapy, nebulisation with bronchodilators and mucolytics, administration
of proteases inhibitors for pulmonary symptoms and administration of substitute
pancreatic enzymes and vitamins for pancreatic insufficiency.
- In advanced stages, the triple (heart-lung-liver) transplant has shown
promising results but the major hindrance is the availability of donor organs.
- Among the latest therapeutic advances, the gene therapy of CF has met
a number of obstacles. Other promising strategies are under study with the
objective of compensating for the defect in the production and/or function
of the CFTR protein depending on the type of mutation.
IV- Diagnosis
- The diagnosis of CF depends on the sweat chloride test. Even today it
is considered to be the most reliable test for CF. The techniques used these
days are simple but the quantity of sweat required is a limiting factor
especially in neonates raising the risk of error to 30%.
- The values of this test can vary according to laboratories; a value of
less than 40 mmoles/l of chloride is generally considered as normal.
- For the range between 40 and 60 mmoles/l, the interpretation is doubtful
and the test should be repeated.
- For the diagnosis to be established, the sweat chloride concentration
must exceed 60 mmoles/l on two separate sweat tests.
- Another technique consists of measuring the bioelectrical potential difference
that exists between the skin and nasal mucosa. It valuable in following
cases:
1. For the early diagnosis in the neonates presenting with a suspected
digestive pathology, where the sweat test is very difficult to be carried
out,
2. For the confirmation of a doubtful diagnosis with some clinical signs
and a borderline or negative sweat chloride test,
3. For the patient follow-up, as there is a correlation between the seriousness
of respiratory involvement measured by FEV1 study (forced expiratory volume
in first second) and the value of bioelectrical potential difference. The
method is simple, less expensive and accessible.
V- CFTR gene and its mutations
V-1. Introduction
CFTR gene, which is responsible for this disorder, contains
27 exons spreading over 250 kb of chromosome 7 (7q31) and encodes an mRNA of
6.5 kb.
V-2. The DF508 mutation
- The most frequent mutation, a deletion of three nucleotides resulting
in the deletion of phenylalanine at position 508 (DF508) is responsible
for 70% of CF alleles.
- There exists a great heterogeneity in the spread of CF, there is a north-west/south-east
gradient, for example 88% of DF508 cases found in Denmark and 50% in Italy.
- To explain the spread of this mutation in European population, the hypothesis
of a selective advantage of heterozygotes was proposed. For example, the
heterozygotes would be protected against dehydration due to diarrhea caused
by enterotoxins of bacteria. The DF508 protein, associated with a decreased
expression of CFTR to the epithelial surfaces, decrease the entry of pathogen
into the intestinal epithelium thus providing a protection against this
infection.
V-3. Spectrum of CFTR gene mutations
- Since the discovery of CFTR gene (http://www.genet.sickkids.on.ca/cftr/)
more than 1200 mutations are described since the cloning of this gene, out
of which 4 (excluding DF508) represent more than 2%. The frequency of certain
mutations can vary among different geographic groups (e.g, W1282X makes
48% of CF alleles among the Ashkenazi Jews and only 2% of the total CF alleles).
- The majority of molecular defects of CFTR gene are the point mutations
out of which 42% are missense mutations, 24% small insertions/deletions
with a frame shift, 16% nonsense mutations, 16% mutations of splicing and
2% deletion of an amino acid. Some major deletions are also reported.
- One of the particularities of CFTR gene is the existence of deleted transcripts
of one or more exons among normal individuals. These transcripts are due
to anomalies leading to alternative splicing out of which the most frequent
and well studied is the deleted transcript of exon 9 (9-). The presence
or absence of this exon is correlated with a "polymorphism" of
sequence of the intron 8 situated near the acceptor site of splicing. Splicing
will occur with 7 or 9T (polypyrimidine tract of intron 8) at about 90%,
but when the length of this sequence is 5T then only 10 to 40% of normal
mRNA will be produced, the rest of 60 to 90% will produce a mRNA without
exon 9 that doesnít produce a functional CFTR protein.
V-4. Genotype-phenotype correlations
- Approximately half of the patients affected by CF are homozygous for
the mutation DF508. DF508 homozygotes present a classical form of the disease
with an increase in electrolytes in sweat, pancreatic insufficiency and
obstructive pathology of lungs.
- The DF508 accounts for 66 % of the mutations, 40 % of these patients
are compound heterozygotes with DF508 on one allele and another mutation
of CFTR gene on the other chromosome.
- Generally, the pulmonary function, the age of onset of the disease, and
the amount of chloride salt are all related to a particular genotype.
- On the other hand, the seriousness and variety of symptoms differing
in the same family explain that the genotype, at the level of CFTR gene,
cannot be solely held responsible for the phenotype.
- Only the mutation A455E was strongly associated with the state of pulmonary
function.
- Concerning the pancreatic function the patients with one or two missense
mutations have a conserved exocrine pancreatic function (PS or pancreatic
sufficiency), as compared to those having two alleles of splicing mutations,
nonsense or frameshift mutations and some mutations of missense which always
lead to pancreatic insufficiency (PI or pancreatic insufficiency). Similarly,
the patients having a PI mutation on one allele and a PS mutation on the
other have a phenotype PS. With a PS mutation, the activity of the CFTR
protein is sufficient for the pancreatic function.
- The effect of one mutation can be modified by a second mutation inherited
in cis on the same allele.
- The polymorphism of the polypyrimidine tract of the intron 8 modifies
the penetrance of certain mutations.
Mutations. Editor.
VI- The CFTR protein and its functions
VI-1. Structure of the CFTR protein
- The protein sequence of the CFTR is composed of 1480 amino acids.
- It consists of two repeated motifs each composed of a hydrophilic membrane-spanning
domain (MSD) containing six helices and an important hydrophilic region
for binding with ATP (NBF or Nucleotide Binding fold). These two motifs
are linked by a cytoplasmic (R domain) encoded by exon 13, containing a
number of charged residues and the majority of the phosphorylation sites
(probable substrates of protein kinases A and/or C).
Figure 1: proposed structure CFTR protein,
by Riordan et al. 1989 - Pascale Fanen.
- Homologies exist between the primary structure of CFTR protein and members
of membrane protein families, the family of ABC transporters (ATP-binding
cassette), which are responsible for the active transport of substrates
across the cell membrane, where ATP hydrolysis serves as the source of energy.
VI-2. Functions of the protein
VI-2.1. Cl- channel function
The early hypotheses regarding the function of CFTR protein
revolved around two possibilities. The first postulated that the CFTR protein
is a Cl- channel. This hypothesis was compatible with the defect in permeability
of Cl- ions at the CF epithelial apical membranes. The other proposed that the
CFTR protein is not an ionic channel but it plays a role in the regulation of
Cl- channel either by associating with them, or by transporting a regulatory
factor for Cl- channels in or out of the cell. The later results determined
the function of CFTR protein as a Cl- channel.
VI-2.2. The CFTR protein, a multifunctional protein
The discoverers of CFTR gene termed it the ëtransmembrane
conductance regulatorí. In fact, the CFTR protein regulates other channels also,
the outwardly rectifying chloride channel (ORCC), epithelial Na+ channel (ENaC)
and at least two inwardly rectifying K+ channels ROMK1 and ROMK2. Besides being
a channel regulator, it also plays a role in transport of ATP, modifying the
phenomenon of exocytosis/endocytosis, regulation of pH of intracellular organelles.
Figure 2: CFTR, a multi functional protein,
by Schwiebert et al. 1999 - Pascale Fanen
VI-3. Correlation of CFTR gene mutations with the Cl- channel
function
The molecular anomalies have variable effects on the CFTR
protein and its functions. Welsh and Smith have proposed a classification of
these anomalies in relation to the Cl- channel function (Welsh & Smith 1993)
(figure 3).
Figure 3: Classification of mutations of CFTR
gene, by Welsh & Smith, 1993 - Pascale Fanen
VI-3.1. Class 1: mutations altering the
production of the protein. These mutations result in the total or partial
absence of the protein. This class includes the nonsense mutations and those
that produce a premature stop codon (anomalies of splicing and frameshift
mutations). In certain cases the mutated mRNA is unstable and doesnít produce
the protein. In other cases, the abnormal protein produced will probably
be unstable and degrade rapidly. This is what produces the truncated protein
or the protein containing the aberrant sequence (anomalies of splicing or
the frame shift). Functionally, these mutants are characterized by a loss
of conductance of Cl- channel in the affected epithelia.
VI-3.2. Class 2: mutations altering the
cellular maturation of the protein. A number of mutations alter the maturation
of the protein and thus the transport of these proteins to the plasma membrane.
In this way, the protein is either absent from the plasma membrane or present
in a very small quantity. The mutations of this class represent the majority
of CF alleles (DF508).
VI-3.3. Class 3: mutations disturbing
the regulation of Cl- channel. These mutations are frequently situated in
the ATP binding domain (NBF1 and 2).
VI-3. 4. Class 4: mutations altering
the conduction of Cl- channel. Certain segments of membrane spanning domains
participate in the formation of an ionic pore. The missense mutations situated
in these regions produce a correctly positioned protein that has a cAMP
dependant Cl- channel activity. But the characteristic of these channels
is different from those of endogenous CFTR channel with a diminution of
ion flux and a modified selectivity.
VI-3. 4. Class 5: mutations altering
the stability of mRNA.
VI-3. 4. Class 6: mutations altering
the stability of mature CFTR protein.
| Written | 09-2001 | Pascale Fanen, Afia Hasnain |
| | Service de Biochimie-Génétique, Inserm U.654, Hôpital Henri Mondor, 94010 Créteil, France |
| This paper should be referenced as such : |
Fanen P, Hasnain A . Cystic Fibrosis and CFTR Gene. Atlas Genet Cytogenet Oncol Haematol. September 2001 .
URL : http://AtlasGeneticsOncology.org/Educ/CistFibID30032ES.html |
|
© Atlas of Genetics and Cytogenetics in Oncology and Haematology | indexed on : Thu Apr 17 13:57:56 2008
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