Biology of Disease

and looking for differences in the sizes of the PCR products. Polyacrylamide

gel electrophoresis (PAGE) can be used to separate the products of PCR of

the small fragment of exon 10 of the CFTR gene and these may be visualized

with ethidium bromide or silver staining. The primers most often used are

called C16B and C16D and generate a 98bp product of exon 10. However, if

the$F508 mutation is present, the PCR product is only 95bp long. This is a

relatively small difference but careful PAGE can easily separate them. This

test is not absolutely specific for $F508 given that other 3bp deletions in this

region would produce the same difference in size. Thus the mutation $I507,

the second identified CF mutation, also gives a 95bp PCR product. Mutations

other than $F508, however, can be identified in many cases because they

produce differing banding patterns following PAGE.

The treatment for cystic fibrosis is aimed at preventing respiratory infections

by regular physiotherapy to try to remove the mucus, along with the use of anti-

biotics to counter bacterial infections (Chapter 3). Pancreatic enzymes, in tablet

form, are given with food to counter the effects of pancreatic insufficiency.

Mice with deletions of cftr, that is gene knockout mice, have been bred to

use as experimental models to test possible therapies. However, such mice

do not develop lung disease in the same manner as humans and therefore

do not provide an effective animal model. This has hampered experimental

gene therapy studies that have tried to insert the normal gene into the lung

epithelial cells of CF patients by using DNA-containing liposomes. To date,

none of these therapies have been successful. Cystic fibrosis used to be fatal

by the age of about 20 years but improvements in treatment mean that many

sufferers now live to be 30 years and over. However, although the prognosis for

CF has improved, many patients still die in early adult life.

16.4 Mitochondrial Disorders

Mitochondria are organelles found in almost all eukaryotic cells although

numbers vary from one to several hundred per cell. They are bounded by

a mitochondrial envelope that consists of outer and inner mitochondrial

membranes with infoldings called cristae, and encloses a central region called

the mitochondrial matrix (Figure 16.6). The inner mitochondrial membrane

X]VeiZg&+/ MEMBRANE, ORGANELLE AND CYTOSKELETAL DISORDERS

W^dad\nd[Y^hZVhZ

Normal

(+/+)

Heterozygote carrier

(+/-)

Homozygote CF

(-/-)

98bp

95bp

Figure 16.5 Detection of the $F508 mutation.

Shows the differences in sizes of the relevant

portions of normal and mutated genes and a

simplified schematic of the separation of these

by polyacrylamide gel electrophoresis (PAGE).

0.3μm

Figure 16.6 Electron micrograph of a

mitochondrion. C, cristae, E, envelope and M,

matrix.

X]VeiZg&+/ MEMBRANE, ORGANELLE AND CYTOSKELETAL DISORDERS

)*+ W^dad\nd[Y^hZVhZ

sequence change. The testing strategy may use both approaches depending

on the purpose of the analysis.

The commonest CF mutation is called $F508. This is the deletion of a codon,

in exon 10 of the CFTR gene, that codes for a phenylalanine (F) residue at

position 508 in the amino acid sequence of the encoded protein. The simplest

way to detect this mutation (Figure 16.5) is by amplifying that part of the gene