Biology Reference
In-Depth Information
structure and function of the protein. Mutations which result in noncon-
servative amino acid substitutions in a part of the protein that is critical to
its function, or in regions known to be conserved between species, are likely
to have significant effects. Examples include a T to G missense mutation in
a family with Pendred's syndrome, which leads to substitution of Cys for
Phe at a highly conserved position in the protein pendrin (
PDS
) (Everett
et al. 1997), and the single nucleotide missense mutations reported in the
POU
homeodomain of the
POU3F4
gene in individuals with the X-linked
form of mixed deafness
DFN3
(Kok et al. 1995).
9.2 Analysis of Control Samples
Supportive evidence that a DNA sequence variant is likely to be a muta-
tion, rather than a normal polymorphism, is provided by the absence of the
sequence variant in an appropriate number of normal control individuals.
The M34T sequence variant in the
Cx26
gene reported in association with
autosomal dominant sensorineural hearing impairment (Kelsell et al. 1997)
occurs in a small but significant proportion of the normal population; this
suggests that it might not be of functional significance, but instead a normal
polymorphic variant (Scott et al. 1998; Kelley et al. 1998). However, in vitro
functional studies (Section 9.3) provide support for the association of this
mutation (101T to C) with hearing impairment.
9.3 In Vitro Functional Studies
In vitro functional assays of a protein can reveal the effect of known or
introduced mutations in a gene. For example, mRNA generated from wild
type and variant
Cx26
gene sequences was expressed in
Xenopus
oocytes
to measure gap junction channel activity. Analysis of the 101T to C
sequence variant in the
Cx26
gene (Kelsell et al. 1997) indicated that it was
likely to be a mutation, rather than a polymorphic variant (White et al.
1998). However, care needs to be taken in the interpretation of the results
of these types of studies, since it is possible that they might not reflect func-
tion in vivo.
9.4 Immunohistochemical/In Situ Hybridization Studies
Further evidence that a mutation in a gene may be responsible for inher-
ited hearing impairment can be obtained by examining the expression of
the gene at the mRNA or protein level in appropriate tissues using
immunohistochemical or in situ hybridization studies. For example,
Cx26
expression is found in the stria vascularis, basement membrane, limbus and
spiral prominence of the cochlea using murine monoclonal antibodies to
Cx26
(Fig. 3.5) (Kelsell et al. 1997).
