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in these genes and then consider how these mutations may act, and whether further such mutations
remain to be found.
Fig. 2. Wild-type and mutant HOXD13. A: Genomic structure of HOXD13, showing sites of reported mutations. D/F= deletion
and frameshift. Exp = expansion. B: Wild-type HOXD13 protein. C and D: Predicted proteins resulting from a 1-bp deletion in the
homeobox (D/F1) and a 14 bp deletion in exon 1 (D/F2), respectively. The former would contain the first 278 wild-type amino acids
followed by 33 novel amino acids, and would lack the last 49 amino acids of the homeodomain. The latter would contain the first
107 wild-type amino acids followed by 115 novel amino acids, and would lack the entire homeodomain. E: Predicted protein
resulting from 7- to 14-residue expansions of the polyalanine tract.
Fig. 3.
Fig. 4. Wild-type and mutant HOXA13. A: Genomic structure of HOXA13, showing sites of reported mutations. X =nonsense
mutation. Exp = expansion. M =missense mutation. B: Wild-type HOXA13 protein. C F: Predicted proteins resulting from the
four nonsense mutations. X1 and X2, both in the homeobox, would remove the last 20 and 24 amino acids respectively, including
three of the four key residues responsible for contacting target DNA in the case of X1, and all four key residues in the case of X2.
X3 and X4, both in exon 1, would remove the last 193 and 253 amino acids, respectively including the entire homeodomain. G:
Mutant HOXA13 protein in the Hypodactyly mouse, caused by a 50-bp deletion in exon 1. The first 25 wild-type amino acids are
followed by 275 novel amino acids. H: Predicted protein resulting from 8-residue expansion of the third polyalanine tract. I:
Predicted protein resulting from missense mutation in the homeobox, which replaces asparagine 51 of the homeodomain by
histidine.
The mutations in HOXD13 and HOXA13 discovered so far fall into three distinct classes: truncation mutations, polyalanine tract expansions and
an amino acid substitution in the homeodomain,
each of which probably acts by a different
mechanism.
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Truncation mutations
The final, and perhaps the most likely, alternative is that the human truncation mutations and
the Hypodactyly mutation all act as null alleles,
and that the targeted mouse mutations have not
produced a straightforward loss-of-function. The
insertion of a selectable marker cassette during the
construction of targeted mouse mutations is
known to be able to disrupt the expression of
neighbouring genes, especially if the genes in question are closely linked or clustered and share regulatory elements, as the HOX genes do (42). This
possibility could be explored by generating mice
carrying targeted HOXD13 and HOXA13 mutations unaccompanied by additional regulatory sequences, using methods that eliminate the selection
cassette, such as the Cre/loxP site specific recombinase system.
Polyalanine tract expansions
As well as being the first human HOX gene mutations to be discovered, the polyalanine tract expansions in HOXD13 that underlie SPD (Fig. 2E)
were also the first instance of a novel class of
mutation, and helped draw attention to the functional importance of HOX proteins N-terminal
regions, hitherto comparatively neglected in favour
of the homeodomain. Similar pathological expansions have subsequently been identified in
HOXA13, as described above (Fig. 4H), as well as
in two non-homeodomain transcription factors,
RUNX2 (formerly CBFA1) (43) and ZIC2 (44),
which result in cleidocranial dysplasia (MIM
119600) and holoprosencephaly (MIM 603073) respectively. (Smaller expansions in PABP2 cause
oculopharyngeal muscular dystrophy, a late-onset
neuromuscular disorder (45), but this protein is not
a transcription factor and the pathological mechanism involved may be different.) In all four transcription factors, the normal polyalanine tracts are
short (1518 residues), and show little or no polymorphism in length, while the expansions are also
short (715 extra residues) and meiotically stable.
They therefore differ sharply from other diseasecausing expansions of trinucleotide repeats, and
are probably caused not by strand slippage but by
unequal crossing-over between two normal alleles
that have become misaligned during replication
(46).
Polyalanine tracts are a common motif in both
homeodomain and non-homeodomain transcription factors, but their normal function is not understood. One possibility is that they act as flexible
spacer elements between other functional domains
(47); another is that they are involved in binding
other proteins with which the transcription factors
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mutation in any one HOXD gene has been excluded, as has a large deletion or chromosomal
re-arrangement in the region (57, 58). In all three
cases, the causative mutation probably affects a
hitherto unidentified cis-acting regulatory element
controlling the entire cluster.
In addition to their role in development, HOX
genes, especially members of the HOXA and
HOXB clusters, are important in normal haematopoiesis. Germline mutations in HOX gene may
therefore cause inherited haematological disorders,
while somatic mutations may play a part in haematological malignancies (59, 60). Recurrent chromosomal translocations producing NUP98/HOXA9
and NUP98/HOXD13 fusion proteins have been
identified in patients with acute myeloid
leukaemia, chronic myeloid leukaemia and
myelodysplastic syndrome (61 65). These fusion
proteins are thought to contribute to leukaemic
transformation by misregulating normal HOX
gene targets in myeloid cells (66). Activation of
HOXA7 or HOXA9 by proviral integration can
lead to myeloid leukaemia in mice (67), as can
overexpression of HOXA9 and HOXA10 in mouse
haematopoietic cells (68, 69). Several DNA-binding partners of the HOX proteins, including members of the PBX and MEIS families, are themselves
implicated in leukaemogenesis (59, 60), and co-activation of MEIS1 and HOXA9 may be common
in myeloid leukaemia (70).
Finally, HOX genes are expressed in many tissues during adult life, including the gut, kidneys,
genital tract and skin, and there is increasing evidence that they are often misexpressed in solid
tumours (71). Somatic HOX gene mutations, particularly regulatory mutations, may therefore have
a wider role in oncogenesis. Interestingly, HOXA5
has recently been shown to activate transcription
of the key tumour suppressor gene p53, and loss of
p53 expression occurs secondary to loss of HOXA5
expression in many human breast cancer cell lines
and tumours (72). This may reflect a somatic lossof-function mutation in HOXA5, but is usually
caused by transcriptional silencing associated with
methylation of the genes promoter region.
HOXA5 may thus itself be an important tumour
suppressor gene, and other HOX genes may prove
to play a similar role in different tumour types. In
cancer as in developmental disorders, the search
for mutations in the human HOX genes may have
only just begun.
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