Biology Reference
In-Depth Information
lamine and osmium tetroxide. Such sites are detected by the addition of
piperidine, which results in cleavage of the DNA at these sites. The tech-
nique identifies mismatches on either strand, and thereby indirectly detects
adenine and guanine mismatches through their complementary nucleotides
on the other strand (Cotton et al. 1988). Chemical cleavage works for longer
DNA fragments than SSCP, DGGE, and heteroduplex analysis and it also
has the advantage that it provides information about the location of the
mutation. Adaptation to fluorescent-based detection systems (Ellis et al.
1998), as well as use of less toxic chemicals (Roberts et al. 1997) and enzy-
matic methods of chemical cleavage (Rzhetsky et al. 1996; Smith and
Modrich 1996; Kortenkamp et al. 1997), may increase the use of chemical
cleavage as a mutation-detection method.
8.2.5 Sequence Analysis
DNA sequencing is the gold standard of mutation detection, but it is expen-
sive and laborious, especially if the gene being sequenced is large. However,
advances in automated DNA sequencing technology are making sequence
analysis the method of choice for mutation detection. In addition, ongoing
developments in DNA chip technology will permit rapid, automated, large-
scale mutation detection once a gene has been identified.
9. Approaches to Confirming that a Detected Mutation
Causes Hearing Impairment
While identification of mutations in affected individuals in a candidate gene
provides strong evidence that it is responsible for inherited hearing impair-
ment, further evidence can be provided in a number of different ways.
9.1 Prediction of the Likely Functional Consequences
of Mutations
Analysis of the functional consequences of specific mutations can pro-
vide compelling evidence that a particular gene is responsible for inhe-
rited hearing impairment. For example, deletion of the whole gene, as has
been reported in some patients with the branchio-oto-renal syndrome
(Abdelhak et al. 1997; Kumar et al. 1998), would be expected to lead to loss
of function. Alternatively, a nonsense mutation, or a frameshift mutation
that generates a stop codon, especially if it occurs in the 5ยข portion of a gene,
leads to a severely truncated protein, which is likely to have severe func-
tional consequences. A number of mutations in the connexin 26 (
Cx26
)
gene are of this type (Avraham and Hasson, Chapter 2).
The likely functional consequence of missense mutations cannot be
predicted reliably in some instances, and may require knowledge of the