2008 Barcelona - European Society of Human Genetics

Clinical genetics
analysed by sequencing exons 1 to 6A . We have found deletions, duplications
or mutations in a total of 10 families . Three of these families
harbour mutations in exon 4, four families had downstream deletions,
and three carry deletions overlapping the SHOX gene . The remaining
family had a duplication upstream of the SHOX gene as the sole trait .
Interestingly, one of the families with a downstream deletion had also
a 5’ duplication of SHOX up to exon 3 that segregated independently .
Deletions and duplications show different breakpoints in each family
supporting the idea that there is no main deletion breakpoint hotspot .
This work has been supported by the grant FIS 05-1585 from the Instituto
de Salud Carlos III from the Spanish ministry of Health.
P01.165
Detection and characterisation of partial SHOX deletions in
patients with Léri-Weill dischondrosteosis (LWD), Langer
mesomelic dysplasia (LmD) and idiopathic short stature (iss)
S. Benito-Sanz 1 , P. Lapunzina 2,3 , N. S. Thomas 4 , Á. Campos-Barros 5 , J. L.
Ross 6 , A. R. Zinn 7 , K. E. Heath 1 ;
1 Hospital Infantil Universitario Niño Jesús, Universidad Autónoma de Madrid,
Madrid, Spain, 2 Hospital Universitario La Paz, Madrid, Spain, 3 CIBERER, Instituto
de Salud Carlos III, Madrid, Spain, 4 Wessex Regional Genetics Service,
Salisbury, United Kingdom, 5 Hospital Infantil Universitario Niño Jesús, Madrid,
Spain, 6 Thomas Jefferson University, Philadelphia, PA, United States, 7 University
of Texas Southwestern Medical School, Dallas, TX, United States.
SHOX is located in the pseudoautosomal region 1 (PAR1) of the X and
Y chromosomes . Mutations in SHOX or in the downstream PAR1 have
been shown to be the cause of Léri-Weill dyschondrosteosis (LWD),
Langer mesomelic dysplasia (LMD) and idiopathic short stature (ISS) .
We routinely perform deletion screening of SHOX and the downstream
PAR1 in LWD, LMD and ISS patients using MLPA . Microsatellite markers
and SNPs are then utilized for further characterisation in specific
cases .
During our screening, we identified nine partial SHOX deletions (five
LWD, one LMD and three ISS) . The deletions were all of variable size,
ranging from the deletion of a single exon to multiple exons . Polymorphisms
in the ligation sites were excluded in cases where deletions
only included one exon . A common 5´ breakpoint region in intron 3 was
observed in five patients. Three patients also share the 3´ limit in intron
2 and two more in the 3´UTR . Deletion breakpoints are currently being
further delimited by fine-tiling CGH arrays and subsequently amplification
across the breakpoints .
MLPA is an accurate, rapid and economic technique to detect complete
and partial SHOX deletions as well as downstream PAR1 deletions
. FISH and microsatellite analysis cannot accurately detect or delimit
this class of deletions . Our results show that, although the partial
deletions of SHOX are variable in size, intron 2 and 3 appear to be
hotspots for breakages .
P01.166
mosaicism of the SHOX gene in a serie of spanish patients with
short stature
L. Magano Casero 1 , K. Heath 2 , P. Lapunzina 3 , P. Arias 3 , I. Incera 3 , S. Benito
Sanz 2 , A. Delicado 3 , R. Gracia Bouthelier 3 ;
1 Hospital Universitario La Paz. Hospital Infantil Universitario Niño Jesús, Madrid,
Spain, 2 Hospital Infantil Universitario Niño Jesús, Madrid, Spain, 3 Hospital
Universitario La Paz., Madrid, Spain.
The SHOX gene, located at the pseudo-autosomic region (PAR1) of
the X (Xp22) and Y (Yp11) chromosomes codifies for a transcription
factor implicated in the regulation of skeletal growth . Deletions or mutations
in SHOX may lead to haploinsufficiency of this gene and sometimes
is associated with Turner syndrome, Leri-Weil dischondrosteosis
(LWD), Langer type mesomelic displasia (LMD) and idiopathic short
stature (ISS) .
PAtiENts AND mEtHODs: Using STRs (microsatellites, repeated
sequences in tandems distributed in the DNA) we evaluated the copy
number of SHOX and/or their different alleles in 340 patients with short
stature . The peak areas of both alleles were evaluated by means of
the ratio between both alleles and then compared and normalized with
normal controls . Calculation was expressed as a percentage of each
allele .
REsULts: We detected mosaicism for SHOX in 5 out of 340 patients .
Normalization showed a percentage of deletion of 46, 33, 26, 26 and
11% respectively . Karyotypes (100 cells were evaluated for each pa-
tient) were normal in all patients .
cONcLUsiON: Though classical haploinsufficiency of the SHOX gene
shows a heterogeneous phenotypic expression in patients with short
stature, patients with mosaicism of this gene should be recognized as
a special group of children since the genetic dose of the gene is variable
and the final height and evolution is unpredictable.
P01.167
Microduplication of the long range SHH limb regulator (ZRS) is
associated with triphalangeal thumb-polysyndactyly syndrome
E. Klopocki 1 , C. E. Ott 1 , N. Benatar 2 , R. Ullmann 3 , S. Mundlos 1,3 , K. Lehmann 1 ;
1 Charite Universitätsmedizin Berlin, Institute of Medical Genetics, Berlin, Germany,
2 Klinik für Handchirurgie, Krankenhaus Marienstift, Braunschweig, Germany,
3 Max Planck Institute of Molecular Genetics, Berlin, Germany.
An important player in establishing the anterior-posterior patterning of
the limb is the developmental regulator gene sonic hedgehog (SHH) .
Previous studies have identified a long range regulator for SHH expression
in the limb bud residing in a highly conserved non-coding
sequence about 1 Mb upstream from the SHH gene itself . As shown
in mice point mutations within this non-coding regulatory region designated
ZRS lead to ectopic expression of Shh in the anterior margin of
the limb bud and thus to preaxial extra digits . In humans ZRS point mutations
are associated with the triphalangeal thumb and polysyndactyly
(TPT-PS, OMIM #174500) phenotype .
In this study we investigated a large pedigree with a variable phenotype
of TPT-PS . Although linkage to the SHH locus was confirmed
sequencing of the ZRS did not reveal point mutations . A subsequent
screening by array-CGH detected a microduplication in 7q36 .3 comprising
the ZRS in an affected individual . The microduplication was
confirmed by qPCR in all affected family members. By using a direct
sequencing strategy we showed that the duplicated segment is in direct
tandem orientation .
In summary we demonstrated that microduplication of the ZRS region
in 7q36 .3 results in a similar phenotype as caused by point mutation in
the limb specific SHH regulatory element . Thus, genomic duplications
have to be considered as a possible mechanism which leads to disturbance
of long-range transcriptional control . The discovery of novel
mechanisms of gene regulation, i .e . distant enhancers/repressors and
their relevance to human disease if disrupted is a challenging task in
the future .
P01.168
The transcription factor TRPS1 interacts with the RINGfinger
ubiquitin ligase ARKADIA
C. Will1 , M. Albrecht1 , S. Gkalympoudis2 , R. Depping3 , G. Gillessen-Kaesbach1 ,
H. Lüdecke2 , F. J. Kaiser1 ;
1 2 Institut für Humangenetik, Lübeck, Germany, Institut für Humangenetik, Essen,
Germany, 3Institut für Physiologie, Lübeck, Germany.
Mutations or deletions of the TRPS1 gene on human chromosome
8q24 .1 cause the tricho-rhino-phalangeal syndromes (TRPS), which
are characterized by craniofacial and skeletal abnormalities . The gene
encodes a transcription factor that functions as a repressor for GATAmediated
transcription . The activity of transcription factors is often
controlled by post-translational modifications. And in fact, we have recently
found that SUMOylation of specific sites within the repression
domain (RD) of TRPS1 regulates its function .
In a yeast-two-hybrid screen we identified two clones encoding amino
acids (aa) 352-505 of the 986 aa protein ARKADIA . ARKADIA is a
RINGfinger ubiquitin ligase. In mice, Arakdia is known as a key regulator
in the TGF-beta pathway by inducing the ubiquitin-dependent degradation
of Smad7, SnoN and c-Ski . By using a variety of truncated
TRPS1 and ARKADIA constructs we could narrow down the ARKA-
DIA-binding region within TRPS1 to the last 100 aa, which includes
the RD . ARKADIA appears to interact with TRPS1 via two different
regions, which were also described to enable the interaction of ARKA-
DIA with SMAD7 . TRPS1 is known to be localized within the nucleus .
Selective inhibition of the proteasome complex with lactacystin results
in cytoplasmic accumulation of TRPS1 in cells co-transfected with
ARKADIA . Furthermore, in luciferase reporter gene assays we could
show that ARKADIA decreases the repressional activity of TRPS1 . Our
results strongly indicate an ARKADIA-mediated ubiquitination which
induces a cytoplasmic degradation of TRPS1 .