Biology Reference
In-Depth Information
function of the nature of the resulting amino acid substitution, its precise location
and immediate environment within the protein molecule, and its effects upon
protein structure and function. Although in-depth investigations of the
in vivo
effects of missense mutations upon specific human proteins are generally rare,
two such studies have nevertheless been performed for human factor IX (Bottema
et al.
, 1991; Wacey
et al.
, 1994), the liver-expressed zymogen of a vitamin K-depen-
dent serine protease that activates factor X in the presence of factor VIIIa. These
studies will be discussed in some detail.
The vast majority of known lesions in the
F9
(Xq26-q27) gene causing hemo-
philia B are missense mutations (Giannelli
et al
., 1996), causing ~59% of severe
(<1% FIX: C) and moderate (1-5% FIX: C) hemophilia, and perhaps as much as
97% of mild (>5% FIX: C) hemophilia (Sommer
et al
., 1992). On the basis of 95
independent missense mutations, Bottema
et al
. (1991) concluded that substitu-
tions of 'generic' factor IX residues (conserved in factor IX of other mammals and
in related human serine proteases) almost invariably cause hemophilia B.
Mutations at factor IX-specific residues (conserved only in mammalian factor IX)
and nonconserved residues were, by contrast, found to be some six to 33-fold less
likely to result in a disease phenotype. Even though the study of Bottema
et al
.
(1991) provided new insights into the identity of amino acid residues of structural
or functional importance to factor IX, the authors did not employ models of the
tertiary structure of the protein or its constituent domains. The significance of
the location of specific amino acid residues within the structure of the factor IX
molecule to the consequences of mutation could therefore not be assessed.
Moreover, neither the variable propensity of different regions of the
F9
gene to
mutate nor the nature of the resulting amino acid exchanges were considered.
In many ways, factor IX represents an ideal system in which to assess the influ-
ence of positional determinants upon the disease-associated mutational spectrum
of a single protein. Firstly, the number of known
F9
missense mutations is among
the highest of all human genes (
Human Gene Mutation Database
;
http://www.uwcm.ac.uk/uwcm/mg/hgmd0.html
). Secondly, the amino acid
sequences of numerous other vertebrate factor IX proteins and evolutionarily
related serine proteases are available for direct comparison (Sarkar
et al
., 1990;
Bottema
et al
., 1991). Finally, the structure of factor IX has been determined by X-
ray crystallography (Brandstetter
et al
., 1995) and the three-dimensional struc-
tures of a number of homologous serine proteases are also known. Wacey
et al
.
(1994) constructed by comparative methods (Swindells and Thornton, 1991), a
multidomain model of the quaternary structure of activated factor IX (FIXa) and
used this model to study the expression pathway of
F9
gene lesions from genotype
to clinical phenotype: a total of 277 different single base-pair substitutions in the
F9
gene, comprising 241 missense mutations and 36 nonsense mutations, were
analysed. Comparison of the relative nearest neighbor-dependent single base-pair
substitution rates in the
F9
gene with estimates derived from a wide range of
other human genes revealed similar profiles (with CpG dinucleotides represent-
ing hotspots for mutation), suggesting that similar mutational mechanisms were
operating at the DNA level.
Wacey
et al
. (1994) classified
F9
missense mutations as either
conservative
or
nonconservative
on the basis of the chemical difference between the wild-type and
