Page 91 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

11.4 Positional Cloning 385

an example the pedigree in Fig. 11.20a. As you can see, the viscous secretions in the lungs, pancreas, sweat glands,
maximum Lod score for these data (which is obtained by and several other tissues. Most cystic fibrosis patients
assuming RF = 12.5%) is 1.1, indicating that this one pedi- die before the age of 30.
gree is insufficient evidence for linkage of the SNP1 locus Positional cloning strategies allowed investigators to
on chromosome 17 and the NF gene. However, if two narrow their search for the causative gene to a 400 kb
additional pedigrees were available, each also with a Lod region between two DNA markers on chromosome 7 that
score of 1.1 (calculated for RF = 12.5%), the Lod score of contained only three candidate genes (previously shown in
the three pedigrees together would be 3.3, constituting Fig. 11.5). One of these genes was CFTR, encoding the
strong evidence that the NF gene is on chromosome 17 and cystic fibrosis transmembrane receptor that allows chloride
linked to SNP1. ions to pass through cell membranes; see Fig. 2.25).
Significantly, both CFTR copies in all cystic fibrosis
patients were found to contain mutations that would alter
Genetic Diseases Can Display Allelic the amino acid sequence of the protein or would prevent
or Locus Heterogeneity normal amounts of the protein from being synthesized.
Thus, as the name implies, CFTR is clearly the gene
Suppose that by positional cloning you have been success- responsible for cystic fibrosis.
ful in narrowing down the location of a disease gene to a One mutation called ΔF508 (which removes the amino
1 Mb long region between two polymorphic markers. In the acid phenylalanine—F—from position 508 of the protein)
human genome, the average gene density is about 1 gene accounts for about two-thirds of all mutant CFTR alleles
per 100 kb of DNA. More than 10 genes might therefore lie worldwide. The remaining alleles consist of more than
in the 1 Mb region. How could you discriminate among 1500 different rare mutations (Fig. 11.23). Many patients
these candidates to find the right one? are thus so-called compound heterozygotes (sometimes
In some cases, it might be possible to find clues by known as trans-heterozygotes), in which one copy of
looking for changes in patients in the amounts or sizes of chromosome 7 has one mutation in CFTR and the other
the mRNA transcripts or the protein products of genes copy of chromosome 7 has a different CFTR mutation. The
(see Problem 38 at the end of this chapter). But by far disease results because neither chromosome 7 can encode a
the most generally useful strategy is to use PCR to am- normal transmembrane receptor; in effect, the two different
plify DNA from all the candidate genes in all available recessive CFTR mutations fail to complement each other.
patients, and then sequence all of these PCR products. If The concept of allelic heterogeneity is central to
you found that the patients all had identifiable mutations understanding a drug that has very recently been developed
in one of these candidate genes, particularly mutations to treat cystic fibrosis effectively, but only in a minority of
that might affect the amino acid sequence of the gene’s patients. In 2012, the United States Food and Drug Admin-
protein product, the evidence would be powerful for istration approved the drug ivacaftor for patients who have
identifying that candidate as the actual disease gene (re- one specific CFTR mutation called G551D (changing
view Fig. 11.18d). glycine at position 551 to aspartic acid). The mutant
Some genetic conditions are always caused by the protein encoded by this allele assembles properly into the
same single mutation in a single gene; we’ve already cell membrane, but the G551D protein is inefficient in
seen that all patients with sickle-cell anemia are homo-
zygous for exactly the same base pair substitution in
the gene encoding the β chain of hemoglobin. DNA Figure 11.23 Allelic heterogeneity in the CFTR gene.
sequencing would thus reveal the same mutation in the Every cystic fibrosis patient has a mutated CFTR gene on both
genomic DNA of all patients and carriers of sickle-cell copies of chromosome 7. The diagram indicates the location of
anemia. mutations at different positions in CFTR relative to the 24 exons
of the gene. Compound heterozygotes are patients who have one
copy of CFTR with one of these mutations, while the other copy of
CFTR has a different mutation.
Allelic heterogeneity: Multiple mutant Exons
14b
14a
alleles in one gene 1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 15 16 17a 17b 18 19 20 21 22 23 24
This simple scenario is not, however, always the case.
Many other genetic diseases display allelic heterogene-
ity, meaning that they can be caused by a variety of dif- Mutations
ferent mutations in the same gene. An important example
is cystic fibrosis, a recessive autosomal genetic condi- In-frame deletion
Missense mutation
tion inherited by 1 child in every 2500 born from two Nonsense mutation
parents of European descent. Children with the disease Frameshift mutation
have a variety of symptoms arising from abnormally Splicing mutation