Download Developmental Mechanisms Underlying Polydactyly

72P
Medical Research Society
M209
Genetic Studies of a Novel Cleft Palate Gene.
MSU Hassan, DT.Bonthron, A F Markhaa
Cleft Palate(CP) is common affecting 1 in 650 newborns. Many
studies confirm a major genetic contribution to the aetiology of CP.
However, only for a small number of rare syndromes have disease
CP-causing mutations been identified. Two lines of evidence suggest
that an important CP locus exists at 2q32-q33. Firstly statistical
analysis of malformations in patients with segmental chromosomal
deletions shows a strong association of CP with this region.
Secondly, our study of two patients who had de novo balanced
reciprocal translocations at t(2;7)(q32;p21) and t(2;l l)(q32;p14)
where CP was their only major malformation. These patients offered
the possibility of homing in on the 2q CP locus by cloning the
(possibly) common breakpoints on chromosome 2.
A contig of PAC clones spanning the region(JOOkb) between the
two breakpoints has been constructed. The sequencing of this region
has now been completed and we have identified only one definite
gene, which encodes a large protein with several DNA-binding
domains that appears to be a transcriptional regulator. I have
identified that this genes ten exons span the entire interval between
the two patients breakpoints, so that this gene's function is disrupted
in both patients. The introns are large, but sequence data has been
obtained allowing PCR-based mutation screening of all coding
exons. Since there are few or no other genes in the breakpoint
interval, I feel it is highly likely that haploinsufficiency of this gene
is indeed responsible for CP in the two patients. Over the last
eighteen months I have designed and optimised methods for
amplifying each exon of the candidate gene and begun to perform
mutation screening in patients with isolated CP. To date, I've
recruited 56 patients and intensive mutation detection on their
genomic DNA samples is well underway. Patient recruitment is on
going. The next stages of my work will involve exploring the
following aspects of my candidate gene: (1) the orthologous murine
gene, which we have now cloned and sequenced, will be inactivated
by homologous recombination in order to explore the effects of
haploinsufficiency in an animal model. (2) the development and
spatial pattern of expression will be determined by in siru
hybridisation. (3) Protein work to define the DNA-binding
specificity and interaction profile to provide insights into the precise
cellular targets for this putative transcriptional regulator.
M210
Developmental Mechanisms Underlying Polydactyly
Miss A Crick,'" J h . JM Brown' and Prof. GM Morriss-Kay'
'Department of Human Anatomy & Genetics, University of
Oxford and *Department of Plastic Surgery, The Radcliffe
Infirmary, Oxford.
The anteroposterior (AP) axis of the limb is characterised in human
and mouse by an anterior biphalangeal digit and four posterior
triphalangeal digits.This AP pattern is thought to be determined by a
diffusible morphogen emanating from a region of posterodistal limb
bud mesenchyme called the zone of polarising activity (ZPA). The
ZPA is defined by its ability to induce digit duplications when
grafted anteriorly into a chick limb bud in ovo. The gene Sonic
hedgehog (Shh)is a good candidate for this putative morphogen:
expression co-localises with the ZPA and ectopic (anterior)
expression has been detected in several mouse mutants with pre-axial
polydactyly. The polydactylous mouse mutant Doublefool (Dbfl is
exceptional in showing normal Shh expression although it has 6-8
triphalangeal digits per limb. However, genes down-stream of Shh
including its own receptor and direct transcriptional target, Patched
( P r d ) , which in the wild-type all have posterior domains of
expression, are all ectopically expressed throughout the distal limb
bud mesenchyme in DbJ Grafts of posterior, mid-distal and anterior
Dbf limb bud mesenchyme into anterior chick limb buds in ovo all
induce digit duplications. Thus, there. is ectopic polarizing activity
and activation of the Shh pathway in the absence of ectopic
expression of Shh itself. Indian hedgehog (Ihh), is another of the
three vertebrate homologues of the Drosophila gene, hedgehog and is
normally associated with patterning of the cartilage anlagen later in
development of the limb. The Ihh locus is within the 0.4cM Dbf
region mapped to mouse chromosome 1. We have shown that Ihh is
precociously and ectopically expressed throughout the distal limb
bud mesenchyme in Dbfand that this pattern of expression correlates
spatially and temporally with the ectopic polarizing activity and
activation of the hedgehog pathway.
Analysis of the developmental mechanisms underlying polydactyly
in this mouse mutant has identified a candidate gene worthy of
investigation in patients with congenital limb abnormalities, the
majority of whom continue to have no genetic diagnosis.
M211
Identifieaton and utilisation of the ORF 73 latency-associated
regulatory region in Herpesvirus saimiri-based vectors
Mathew Giles, Peter Smith,
Whitehouse
Kersten Hall and Adrian
Molecular Medicine Unit, University of Leeds, St. James's
University Hospital, Leeds, LS9 7TF
Herpesvirus saimiri (HVS) is an attractive candidate for use as a
gene therapy vector. HVS can efficiently infect a wide variety of
human carcinoma cell lines without cytopathic effects. Moreover, the
HVS viral DNA is clearly able to establish a latent episomal state
within human cells and segregate to progeny upon cell division, a
combination of characteristics which is not possessed by any other
herpesvirus. At present, the first generation HVS vectors utilize
heterologous promoters such as the CMV immediate early
promoters. However, these promoters maybe unsuitable for long
term expression in vivo, as promoter silencing has been observed in
other herpesvirus-based vector systems. Therefore, alternative
promoters to maintain long term expression in HVS vectors are
required. Ideal promoters would function in a viral latent state.
Here we describe the identification of a cluster of genes encoding
ORF 71-73, which are latently expressed in an A549 cell line stably
transduced by HVS-GFP. Moreover, these genes are transcribed as a
polycistronic mRNA species from a common latency-associated
promoter upstream of ORF 73. Utilising deletion analysis we have
identified the minimal latency-associatedpromoter which we believe
will be sufficient to drive long term therapeutic gene expression in
HVS-based vectors. To test this hypothesis we have produced a
recombinant HVS which incorporates the minimal latency-associated
promoter elements expressing GFP.Upon infection of a wide range
of carcinoma cell lines, the recombinant virus remains as a circular
non-integrated circular episome allowing expression of the transgene
from the latency-associated promoter. Furthermore, human cancer
cell xenograft experiments have demonstrated that this promoter is
functional in an in vivo environment. Therefore, we believe that the
ORF 73 promoter is an ideal choice of regulatory sequence for
driving stable long term expression of a transgene in HVS-based
gene delivery vectors.