Chris Bennett and Daniel Peckham. August 2002. The genetics of cystic fibrosis [online]. Leeds University Teaching Hospitals, Leeds, UK. Available from http://www.cysticfibrosismedicine.com
The nucleus of every human cell contains forty-six chromosomes that are made up of long coils of double stranded DNA. Forty-four of these chromosomes are matched into twenty-two pairs and are numbered from 1 to 22 (autosomes). The last pair makes up the sex chromosomes, X or Y. Females receive two X chromosomes while males receive an X chromosome from their mother and a Y from their father.
The chromosomes contain around 35,000 genes. These genes are made up of segments of DNA and are the body’s blueprint for the manufacture of proteins. The latter are the body’s essential building blocks. Each gene comes in as a pair, one inherited from the mother and the other from the father. The exception being those genes on the X chromosome.
The CF gene
Over recent years there has been a dramatic increase in our understanding
of the genetics of cystic fibrosis (CF). In 1985 the gene was localised
to 7q21-34 on the long arm of chromosome 7 (Wainwright et al, 1985)
and this was soon followed by identification of the gene sequence (Rommens
et al, 1989; Riordan et al, 1989, Kerem et al, 1989). The gene encodes
a 1480 amino acid protein, which has been named the cystic fibrosis transmembrane
conductance regulator or, for short, CFTR.
Incidence
Cystic fibrosis remains one of the commonest life threatening autossomal recessive condition affecting Caucasians. The incidence is 1 / 2,500–1 / 90,000, varying between populations. In the UK, the incidence is about 1 / 2,500 live births and the carrier frequency is 1/25. It is uncommon in Asians and Africans. The high prevalence of the CF gene in certain populations has led to speculations that there may be some heterozygotic advantages (Gairdner D, 1975; Pritchard, 1991; Jorde 1988). It has been postulated that carrier status may be linked to improved survival (less chloride loss with the diarrhoea) following infections such as cholera and typhoid. The evidence is conflicting.
Mode of inheritance
Cystic fibrosis has a simple Mendelian autosomal recessive inheritance (figure 1). Affected individuals will have two copies of the mutated CFTR gene, one inherited from each parent. Carriers will have one normal and one mutated CFTR gene and their health will not be affected. However carriers will have the potential to pass on the gene to their offspring. Brothers and sisters of affected individuals are at increased risk (1 in 4) of having CF because both parents will be carriers.
If two carriers of the mutated CF gene have children then there is:
• A one in four
chance that their baby will have CF
• A one in four chance that their baby will not have CF or carry
a CFTR mutated gene
• A two in four chance that the baby will not have CF but will carry
one CFTR mutated gene
What is CFTR?
CFTR is a protein that is found in various cell types, including lung epithelium, submucosal glands, pancreas, liver, sweat ducts and reproductive tract. It comprises two membrane-spanning domains and two nucleotide-binding domains separated by a regulatory R domain. The two membrane-spanning domains form a low-conductance chloride channel pore (figure 2). This is regulated by the binding and hydrolysis of ATP at the nucleotide-binding domains following initial phosphorylation of the R domain. CFTR also regulates ion transport. It has inhibitory effects on apical sodium permeability across epithelial surfaces and activates non-CFTR chloride channels
Ion transport
The widespread presence of CFTR throughout the body (the lungs, salivary glands, pancreas, liver, sweat ducts and reproductive tract) helps to explain why CF is a multisystem condition affecting many organs (figure 3). The two major systems affected, however, are the lungs and the gastrointestinal tract.
In the lungs, impaired CFTR function causes defective chloride transport across the apical membrane and enhanced sodium absorption through epithelial Na+ channels (ENaC) and basolateral Na/K ATPase pumps. These changes in ion transport lead to a net increase in water absorption, a reduction is the depth of airway surface liquid and impaired ciliary clearance. Impairment of the CFTR protein has also been linked to defective bacterial adhesion and reduced clearance and phagocytosis (destruction) of bacteria, such as P. aeruginosa.
figure
4: In cystic fibrosis, the lack of CFTR function caused reduced fluid
production and enhanced sodium absorption through epithelial Na+ channels
(ENaC) and basolateral Na/K ATPase pumps. This results in increase fluid
absorption leading to dryer airways and impaired ciliary clearance.
When the delta F508 gene was identified in 1989 clinicians hoped that genetic testing would provide a sensitive and specific diagnostic test. Unfortunately this has not proved to be the case. In most cases if a patient has two known CF mutations then this confirms the diagnosis. There are, however, more than 1000 different CF mutations and in routine practice it is only possible to check for approximately 100 of these. For an up-to-date register consult the world wide web site at http://www.genet.sickkids.on.ca/cftrl. It is therefore possible to confirm the diagnosis of CF by means of the genotype but not possible to exclude it. There are some mutations which may not be enough alone to confirm the diagnosis.
Class of CFTR mutation
More than 1000 cystic fibrosis mutations have been reported, many of which are rare. The various mutations affect the function of CFTR in different ways Some result in abnormal CFTR production and others affect the intracellular processing of CFTR, channel function or a combination of these (Zielenksi & Tsui, 1995).
The most common mutation worldwide is DF508, which occurs in 75% of cystic fibrosis patients in the UK. A three base-pair deletion in exon 10 results in the omission of phenylalanine at position 508 of CFTR, leading to a combination of defective intracellular processing (which results in an absence of CFTR from the membrane) and defective channel function. In other ethnic groups the range of mutations are different eg in Ashkinazi Jews DF508 is 27% and W1282X 51% of the mutations (Kalman et al, 1994).
It is assumed
that nonsense, frameshift and splice site mutations result in the absence
of functional CFTR. Missense mutations are more problematical in that
they may only partially affect CFTR function. As the amount of functioning
CFTR appears to be related to clinical status, some ‘milder’
mutations may present with congenital absence of vas with or without mild
respiratory disease (table 1) (Massie et al, 2001; Davis, 1996).
| Percentage
of normal CFTR function |
Manifestations
of Cystic Fibrosis |
<
1 % |
classic
disease |
<
4.5 % |
progressive
pulmonary disease |
<
5 % |
clinically
demonstrable sweat abnormality |
<
10 % |
congenital
absence of the vas (male infertility) |
10-49
% |
no
known abnormality |
50-100
% |
no
known abnormality (asymptomatic heterozygotes) |
There are various mechanisms by which mutations in the CF gene produce quantitative or qualitative changes in CFTR function. Five functional classes of CF mutations are described, (Zielenksi & Tsui, 1995).
Class
of mutation |
CFTR production and function |
Class
1 mutations |
Defective
protein production with premature termination of CFTR production.
Class 1 mutations produce few or no functioning CFTR chloride channels |
Class
2 mutations |
Defective trafficking of CFTR so that it does not reach the apical
surface membrane where it is intended to function |
Class
3 mutations |
Defective
regulation of CFTR even though it is able to reach the apical cell
surface |
Class
4 mutations |
CFTR
reaches the apical surface but conduction through the channel is
defective |
Class
5 mutations |
Associated
with reduced synthesis of functional CFTR |
Phenotypic expression
Since sweat testing was introduced there have been individuals who are atypical. They have features of cystic fibrosis but a normal or borderline sweat test. This includes mild lung disease (Gan et al, 1995), pancreatitits (Cohn et al, 1998) and bilateral congenital absence of the vas deferens (CAVBD) (Dumur et al, 1990).
This has lead to the recognition that the spectrum of mutations in the CFTR gene gives rise to a very variable clinical phenotype that may not be predictable from the genotype.
Some mutations may act concurrently with other mutations on the same allele. An example of this is exon 9 splicing which is influenced by the polythymidine sequence of intron 8 which precedes the splicing receptor site (Massie et al, 2001). This polythymidine tract is polymorphic with sequences of 5, 7, 9 thymidines. Because CFTR missing exon 9 splicing is non functional and exon-9 splicing is inversely proportional to the length of the thymidine sequences, the 9T variant allows normal reading of the gene while the 5T variant is associated with the highest level of non-functional CFTR protein (Massie et al, 2001).The commonest polymorphism is the seven thymidine (7T) variant and the DF508 mutation occurs exclusively on the 9T variant. This variation may be important in some CFTR mutations eg R117H . The presence of R117H/508 on a background of 5T is associated with an elevated sweat test and clinical CF. R117H in association with 7T is associated with a normal, borderline or elevated sweat test and variable clinical presentation. (Massie et al, 2001; Rave-Harel et al, 1997; Kiesewetter et al, 1993).
Environment factors and the influence of modulator genes are also likely to impact on phemotypic expression..
Use of DNA testing
The identification of the CFTR has allowed the diagnosis of cystic fibrosis to move into the realm of molecular diagnosis. Most DNA diagnostic laboratories will screen for the commoner mutations
Mutation |
Prevalence
(%) |
DF508 |
79 |
G551D |
2.17 |
R117H |
0.7 |
621+1
(G>T) |
0.5 |
G542X |
0.5 |
N1303K |
0.35 |
1717-1
(G>T) |
0.28 |
R1162X |
0.14 |
R553X |
0.14 |
3849+10KB
(G>T) |
0.07 |
R334W |
0.07 |
W1282X |
0.07 |
TOTAL |
84 |
The screening of CFTR mutations can be used in the diagnosis of CF. The presence of two mutations known to cause CF indicates that the individual has CF. The finding of only one mutation or no mutations does not exclude the diagnosis.
Sample types
The laboratory will be able to test a variety of samples eg saliva, dried blood spots, blood and solid tissue. The usual sample is 5-10mls of blood in EDTA. Unless there are already arrangements to use other tissue then the type of sample should be discussed with the laboratory
Screening Siblings
Generally the major risk for recurrence of CF in succeeding generations is to siblings of known affected individuals, the major exception being consanguineous families.
If both mutations are known in a person with CF this information can be used to predict whether siblings are affected or not.
If no mutations have been identified but the diagnosis is not in doubt, using samples from the affected individuals and parents we can identify which of the parental chromosome 7’s segregate with the disease. If the parents have passed the same two chromosome 7‘s to another offspring then they are considered to be affected (Brock et al, 1998).
Population Screening
Neonates screening positive for cystic fibrosis using immunoreactive trypsinogen measurements on dried blood spot samples can be subsequently screened for CFTR mutations Those carrying two mutations can be diagnosed as having CF. Those having only one mutation should have two sweat test.
It is also possible to screen the population to identify couples at risk of having children with cystic fibrosis and they may wish to undertake prenatal diagnosis. This approach will not detect all carriers.
Prenatal diagnosis.
The diagnosis of cystic fibrosis can be made during pregnancy as long as the CF mutations are known. Samples are obtained by chorionic villous sampling (CVS) or amniocentesis.
Ultrasound examination of the foetus may detect foetal echogenic bowel. One association of this is with cystic fibrosis.
In some specialist
fertility units preimplantation genetic diagnosis (PGD) can be undertaken
successfully (Vrettou et al, 2002; Harper et al,2002; Ray et al, 2002).
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