2008 Barcelona - European Society of Human Genetics

Clinical genetics
P01.160
characterization of the Exostosin-1 (EXt1) promoter
I. Jennes1 , W. Wuyts1,2 ;
1 2 University of Antwerp, Antwerp, Belgium, University Hospital of Antwerp, Antwerp,
Belgium.
Introduction
Multiple osteochondromas (MO) is an autosomal dominant skeletal
disorder characterized by the formation of multiple cartilage-capped
protuberances .
MO is genetically heterogeneous and is associated with a mutation in
the EXT1 or EXT2 tumour suppressor genes . Both genes are ubiquitously
expressed and encode proteins that function as glycosyltransferases
in the biosynthesis of heparan sulphate .
At present, very little is known about the transcriptional regulation of
the EXT genes . To elucidate transcriptional regulation of EXT1, we
isolated and characterized the EXT1 promoter .
Methods and results
Theoretical analysis of the 10 kb upstream of the EXT1 start codon was
performed with promoter prediction programs TSSG, TSSW, FPROM,
BDGP, Promoter 2 .0 Prediction Server and Web Promoter Scan . This
showed presence of a CpG island containing CG and CAAT boxes but
no TATA box which, characteristic for a housekeeping gene . Two potential
functional promoter regions were identified located respectively
~2 .650 bp and ~900 bp upstream of the start codon .
Overlapping PCR fragments of the 10 kb putative EXT1 promoter region
were generated and cloned in the pGL4 .72 Luciferase Reporter
Vector . Promoter activity was determined by luciferase assays after
transfection in Human Embryonic Kidney (HEK) cells . This situated
the actual core promoter within the fragment containing the predicted
sequence ~900 bp upstream of the start codon. Further fine mapping
of the core promoter and potential regulatory sites was achieved by
generating additional subclones, which were subsequently analyzed
for promoter activity and analyzed for protein binding capacities . This
allowed identification of EXT1 promoter binding proteins.
P01.161
mutation screening for autosomal recessive malignant
osteopetrosis
S. Akbaroghli 1 , T. Majidizadeh 1 , M. Dehghan Manshadi 1 , M. Rostami 1 , M. Nateghi
1 , M. Sanati 2 , M. Houshamnd 2,1 ;
1 Special Medical Center, Tehran, Islamic Republic of Iran, 2 National Institute of
Genetic Engineering & Biotechnology, Tehran, Islamic Republic of Iran.
Osteopetrosis is a congenital disorder characterized by defective or
absent osteoclasts, the cells that break down bone . In healthy bone,
a balance is achieved between the production of bone by osteoblasts,
and the break down of bone by osteoclasts . In osteopetrosis, osteoclasts
don’t function normally, and the production of bone by osteoblasts
leads to bones that are abnormally dense and brittle . Osteopetrosis
may result from conditions which interfere with the production of
osteoclasts and their ability to remove bone .
Several forms of osteopetrosis exist . The most common form is autosomal
dominant . The most severe form of osteopetrosis is termed
infantile osteopetrosis, as affected individuals usually have difficulties
soon after birth, inherited in an autosomal recessive pattern . The disease
is very rare, with about 1/300,000 children having severe osteopetrosis
. The candidate genes for Osteopetrosis are TCIRG1, CLCN7
genes . The spectrum of CLCN7-related osteopetrosis includes infantile
malignant CLCN7-related autosomal recessive osteopetrosis,
intermediate autosomal and autosomal dominant osteopetrosis type
II . Mutations of the TCIRG1 gene are the most frequent cause of AR
osteopetrosis . TCIRG1 encoding the a3 subunit of the vacuolar proton
pump is located on chromosome 11q13 consists of 20 exons .
In present study DNA was received from a consanguineous couple
who had 1 affected child from who no sample was available and they
were referred us for screening the problem if any, in the fetus . For each
sample we performed genetic analysis of hot spot axons of TCIRG1
by direct sequence analysis . We reported a new mutation in TCIRG1
gene .
P01.162
A novel homozygous cOLL11A2 deletion causes a c-terminal
protein truncation without mRNA decay in a turkish patient
O. Z. Uyguner1 , H. Kayserili1 , G. Guven1 , M. U. Emiroglu1 , N. Baserer1 , B.
Wollnik2 ;
1 2 Istanbul University, Istanbul, Turkey, University of Cologne, Cologne, Germany.
Otospondylomegaepiphyseal dysplasia (OSMED, OMIM 215150) is a
rare autosomal recessive disorder of bone growth and development
that results in disproportionate shortness of the limbs with abnormally
large knees and elbows, severe hearing loss, and distinctive facial
features presenting mid-face hypoplasia, depressed nasal bridge with
anteverted nares . Cleft palate and micrognathia are also the common
findings. The phenotype is very similar to non-ophthalmic Stickler or
Stickler Type III (OMIM 184840), which displays more subtle signs .
Recessive mutations on collagen peptide coding gene, COL11A2, are
responsible for OSMED while dominant mutations are associated with
Stickler Type III . Furthermore, mutations in the COL11A2 gene have
been also found in patients with isolated cleft palate, Robin sequences,
micrognathia, and non-ophthalmic Stickler syndrome . In this study, two
cousins with OSMED were clinically assessed and mutations testing
on patient’s cDNA identified the novel homozygous c.2763delT in exon
38 of the COL11A2 gene . The deletion which causes a frame shift after
position 425 and a premature stop after additional 62 amino acids
(p.P425PfsX62) was confirmed on genomic level in both patients and
was not found in 200 ethnically matched control chromosomes .
P01.163
mutations of the sequestosome 1 gene associated with Paget‘s
disease in patients from salamanca, spain
E. Corral Moro 1 , L. Corral Gudino 2 , J. García Aparicio 2 , S. Ciria Abad 1 , N.
Alonso López 1 , J. del Pino Montes 2 , R. González Sarmiento 1 ;
1 Unidad de Medicina Molecular-Departamento de Medicina, Universidad de
Salamanca, Salamanca, Spain, 2 Servicio de Reumatología, Hospital Universitario
de Salamanca, Salamanca, Spain.
Paget´s disease of bone (PDB) is a common condition characterized
by focal abnormalities of increased bone turnover .
Mutations in the sequestosome 1/p62 gene (SQSTM1) are associated
with Paget linked to the 5q35 .
Mutations described to date cluster within the C- terminal ubiquitin-associated
(UBA) domain of p62 and patients with truncating mutations
of p62 in or close to the UBA domain generally suffer more severe
disease than those with missense mutations .
We investigated a cohort of 20 familial PDB patients for sequestosome
mutations .
We identificated three mutations within exon 8: P392L, M404T, previously
reported by other groups, and A426V ,a novel missense mutation
. All three of these mutations affect the ubiquitin-associated (UBA)
domain of the protein which is involved in the ubiquitin binding .
Moreover, analysis of exon 6 detected a G to C substitution at position
+822, resulting in a glutamic acid to aspartic acid substitution at codon
426 . This mutation affects the PEST sequence, important for the span
life to the protein . Actually, more studies are under way to examine the
effect of his mutation .
P01.164
mutations, duplications and deletions, upstream, downstream
and overlapping the sHOX gene in Leri-Weill dyschondrosteosis
or short stature patients of the Balearic islands
J. Rosell1 , J. Ferragut2 , M. Caimari2 , N. Govea1 , M. Bernues1 , A. Perez-Granero1
, D. Heine-Suñer1 ;
1 2 Genetics Hospital Universitari Son Dureta, Palma de Mallorca, Spain, Paediatrics
Hospital Universitari Son Dureta, Palma de Mallorca, Spain.
The SHOX gene is located on the pseudoautosomal regions (PAR1)
of the X and Y chromosomes . Mutations and deletions of SHOX have
been shown to be associated with Leri-Weill dyschondrosteosis (LWD)
and idiopathic short stature (ISS) . Recently, it has been shown that deletions
downstream within in the PAR1 region and that do not include
the SHOX gene can be also associated with LWD . We have studied a
total of 120 ISS and LWD patients and their families from the Balearic
islands population . The presence of deletions has been determined
using microsatellites that are located upstream and/or MLPA (SALSA
18C) which can detect downstream deletions . Mutations have been