Biology of Disease

cyclic AMP-regulated chloride channel and has been identified, cloned and
sequenced and most of its mutations are known. The decreased permeability
results in many exocrine secretions with altered ion and reduced water con-
tents and high viscosities. The appropriate viscosity is essential for normal
functions of lubrication, digestion and protection by proteins secreted at
epithelial surfaces. The highly viscous secretions can cause obstructions in
ducts and lead to an accumulation of mucus and colonization by bacteria.
Considering that CF is essentially a fatal disease, one may wonder why the
mutated genes persist in the population. One theory is that mutations in
CFTR increase resistance to cholera (Chapters 2and 3 ). The toxin of Vibrio
cholerae (Figure 16.4) binds to surface receptors on the plasma membranes
of gut enterocytes, leading to a voluminous secretion of fluid and Cl– result-
ing in a secretory diarrhea that can be fatal if untreated. Cystic fibrosis mice
that do not express cftr do not secrete fluid in response to cholera toxin.
Heterozygous CF mice that express half the normal amount of cftr protein
secrete about half the normal fluid and Cl– in response to cholera toxin.
Thus CF heterozygotes might have a selective advantage in being resistant
to cholera.

Diagnosis and treatment of cystic fibrosis

A diagnosis of CF can be confirmed by demonstrating a concentration of Cl–
in sweat higher than 70 mmol dm–3. Pilocarpine is first applied topically to
patients to stimulate the flow of sweat, samples of which are then collected
and analyzed for Cl–.

A number of screening tests for CF are now available based on the effects of
CF on pancreatic function. The most popular of these relies on measurement
of immunoreactive trypsin (IRT) in dried blood spots, similar to those col-
lected for PKU (Chapter 15). Immunoreactive trypsin is greatly increased in
infants with CF in the first month of life compared with that of healthy infants.
However, the test cannot be used after the first few weeks of life since IRT
levels fall as pancreatic insufficiency develops.

The identification of mutations in the CFTR of a cystic fibrosis patient
will confirm the diagnosis and it can also assist with carrier testing of the
relatives of the patient. It is also used to give an accurate and rapid prenatal
diagnosis of CF once the parental genotypes have been confirmed. Currently
there are over 1300 mutations listed in the CFTR mutation database with
the largest number (500) being attributed to missense mutations. When it
is not possible to detect mutant CFTR alleles, then the sweat test remains
the definitive test for diagnosis of CF but, unfortunately, the sweat test is
not always reliable in the first six weeks of life or in adulthood. Furthermore,
some patients with CF, including a number with severe lung disease, give a
normal sweat test.

The techniques for genetic analysis of CF are based on the detection of an
alteration in the DNA sequence of the CTFR gene. In principle, any method
capable of detecting a change in the DNA sequence can be used in mutation
detection. Current techniques rely in one way or another on the hybridization
of two single strands of DNA and on the ability of changes in the sequences
of DNA to alter the kinetics of hybridization. These techniques depend on
the polymerase chain reaction or PCR (Chapter 3) and this, in turn, requires
that the normal sequence of the gene, including its intron–exon boundaries
(described in Chapter 13) be known, as is the case for CF. Polymerase chain
reaction primers are designed that will bind to adjacent regions of the gene
of interest and allow it to be amplified. In most cases, whole exons are ampli-
fied together with a short region of their neighboring intron sequences to give
a PCR product 300 to 500 base pairs long. These products are then analyzed
for specific point mutations or for generalized, nonspecific evidence of a

PLASMA MEMBRANE DISORDERS

CZhhVg6]bZY!BVjgZZc9Vlhdc!8]g^hHb^i]:YLddY )**

1.4 μm

Figure 16.4 Light micrograph of Vibrio cholerae.

Courtesy of Public Health Image Library, Centers for

Disease Control and Prevention, USA.