2009 Vienna - European Society of Human Genetics

Laboratory and quality management
P14.21
Higher efficiency of TA-clamp modified single stranded
oligonucleotides in targeted nucleotide exchange is not
correlated to a lower intracellular degradation
M. Wuepping, D. Kaufmann;
Institute of Human Genetics, Ulm, Germany.
Specific single stranded oligonucleotides can induce targeted nucleotide
sequence correction in an eukaryotic genes in vitro and in vivo.
Our model for investigating the reasons for the low correction rates
achieved with this method is the correction of a point mutation in the
hypoxanthine-guanine-phosphoribosyl-transferase (hprt) gene in the
cell line V79-151. Using single stranded phosphorothioate modified
oligonucleotides the correction rates of this hprt mutation were low but
always reproducible. One reason for low exchange rates may be a
very fast intracellular degradation of the oligonucleotides. Therefore
we compared the exchange rates of different 3´- and 5´-end modified
oligonucleotides with their degradation rates. TA-repeat (clamp)
modified oligonucleotides showed higher correction rates than those
with a GC-clamp and 5´-clamps induced higher correction rates than
clamps at the 3´-end. Experiments on the stability of the most effective
5´-TA and 3´-TA-clamp modified oligonucleotide indicated a very rapid
cleavage and the occurrence of shortened oligonucleotides in the
presence of cytoplasmic and nuclear extracts. The phosphorothioate
modified oligonucleotides were more stable, but their correction rates
were lower. We suggest that there is no direct correlation between the
biological stability of the full length oligonucleotides and the exchange
rates achieved.
P14.22
Rapamycin treatment of a girl with double phakomatosis:
tuberous sclerosis and neurofibromatosis type 1
K. Mayer 1 , H. Seidel 2,3 , A. Wiemer-Kruel 4 , I. Rost 1 , M. Staehler 5 , M. Fischereder
6 ;
1 Center for Human Genetics and Laboratory Medicine, Martinsried, Germany,
2 Institute of Human Genetics, Technical University, Munich, Germany, 3 Institute
of Human Genetics, Ludwig Maximilians University, Munich, Germany, 4 Epilepsy
Centre Kork, Clinic for Children and Adolescents, Kehl-Kork, Germany,
5 Department of Urology, Ludwig Maximilians University, University Hospital
Grosshadern, Munich, Germany, 6 Department of Nephrology, Medical Policlinic,
Ludwig Maximilians University, Munich, Germany.
Tuberous sclerosis (TSC) and neurofibromatosis type 1 (NF1) represent
the two most common neurocutaneous disorders. Both are autosomal
dominantly inherited with well-delineated genetic and clinical
findings. However, the simultaneous presentation in one patient is quite
rare. We report a 13 year old girl with the clinical diagnosis of TSC including
skin, brain and kidney lesions and additionally an increasing
number of cafe-au-lait spots. Molecular analysis revealed a de novo
TSC2 mutation and a NF1 mutation inherited from the mother.
The gene products of TSC1 and TSC2, hamartin and tuberin, respectively,
form a complex and inhibit the mammalian target of rapamycin
(mTOR) in the correspondent signalling pathway. The drug rapamycin
has been shown to suppress mTOR signalling which is activated in
renal angiomyolipoma due to TSC mutations. The patient has been
treated with rapamycin for six months in order to reduce angiomyolipoma
volume prior to organ preserving renal surgery. The achieved significant
reduction of tumour volume allowed partial nephrectomy and
complete AML resection. As an additional benefit, cognitive functions
improved as a consequence of decreased epileptic activity.
NF1 is caused by loss-of-function mutations of the NF1 gene encoding
neurofibromin, a RasGAP. In NF1 deficient human tumours, Ras
and mTOR are activated through cross-talk of Ras/MAPK and PI3K/
Akt/mTOR signalling pathways. To our knowledge this is the first patient
with TSC and NF1 treated with rapamycin. Although she currently
presents only cafe-au-lait spots potential future rapamycin treatment
focussing on NF1 lesions will show if the drug also exhibits a viable
therapy for NF1.
P14.23
Particularity of the therapy in a case with turner syndrome
associated with juvenile rheumatoid polyarthritis
M. Cevei1 , D. Stoicanescu2 , D. Farcas1 ;
1 2 Faculty of Medicine and Pharmacy, Oradea, Romania, University of Medicine
and Pharmacy, Timisoara, Romania.
Juvenile rheumatoid arthritis is the most common type of childhood
arthritis. It is an autoimmune, chronic disease that most commonly
causes inflammation and tissue damage in joints and tendons. The
most common features are: joint inflammation, joint contracture, joint
damage and/or alteration or change in growth. Other symptoms include
joint stiffness following rest or decreased activity level and
weakness in muscles and other soft tissues around involved joints.
Turner syndrome is a chromosomal disorder with characteristic physical
abnormalities, such as short stature, signs of ovarian failure and
also skeletal dysplasia. We present the case of an 18 years old girl
diagnosed with juvenile rheumatoid arthritis at the age of 3 years. During
childhood she was also diagnosed with Turner syndrome. Besides
replacement therapy, she was initially treated with nonsteroidal antiinflammatory
drugs and prednisone and then with methotrexate and
Enbrel. Treatment focused on preserving physical activity to maintain
full joint movement and strength, preventing damage and controlling
pain. Medical rehabilitation treatment was associated to the biological
therapy, the main objective being maintaining mobility and functional
parameters for an active life. As there is an association of these two
conditions, the standard therapy could not be applied, she could not
receive the efficient doses of drugs, and the rehabilitation program has
been modulated according to the existing statural deficit and coexistence
of modified joints with limited range of motion.
P15. Laboratory and quality management
P15.01
the POcEmON (Point-Of-care mONitoring and Diagnostics for
Autoimmune Diseases) project: building a Lab-On-chip centered
on Rheumatoid Arthritis and multiple sclerosis.
S. Lupoli 1,2 , C. Cosentino 2 , V. Tieran 2 , F. Taddeo 2 , S. Atkinson 3 , A. Barton 4 ,
A. Wilson 3 , D. Plant 4 , J. Maxwell 3 , I. Chumakov 5 , F. Kalatzis 6 , K. Schicho 7,8 , H.
Gruessinger 8 , F. Macciardi 2 ;
1 INSPE, HsR Scientific Institute, Milan, Italy, 2 University of Milan, Milan, Italy,
3 University of Sheffield, Sheffield, United Kingdom, 4 arc Epidemiology Unit,
University of Manchester, Manchester, United Kingdom, 5 Pharnext S.A., Paris,
France, 6 Unit of Medical Technology and Intelligent Information Systems,
University of Ioannina, Ioannina, Greece, 7 Medical University of Vienna, Cranio-Maxillofacial
and Oral Surgery, Vienna, Austria, 8 PCS Professional Clinical
Software GmbH, Klagenfurt, Austria.
POCEMON is a large-scale integrated project founded from the European
Commission (FP7-ICT-2007-216088). The aims of the project
are to develop an integrated diagnostic platform mainly dedicated to
Rheumatoid Arthritis (RA) and Multiple Sclerosis (MS) in a first stage
by combining Lab-On-Chip technology, DNA microarray genotyping,
microelectronics, mobile devices, intelligent algorithms and wireless
communications.
MS and RA are two progressive autoimmune diseases and are causes
of disability in young adults. Considering the social relevance of MS
and RA, it is extremely important to improve the timing and confidence
of the diagnosis. According to this vision the POCEMON project can be
a real milestone for the improvement of life style of many patients.
In a first (discovery) phase, we are performing a case/control whole
genome association study, mostly centered to identify HLA (Human
Leukocyte Antigens) and other potentially relevant susceptibility genes
for the two diseases. For the discovery phase of RA, we are using a
homogeneous North-European population (with 800 cases and 800
controls). The discovery is then followed by a confirmatory phase,
where the “best” SNPs are evaluated in a second independent sample
(2000 cases affected by RA or MS respectively and relative controls),
from three separate cohorts.
The genotyping phase for RA, using Illumina HumanCNV-370, is concluded.
Plink 1.05 is used for QC of genotyping data, single marker
association analysis and permutations. The analysis is ongoing. Initial
findings pointed to several susceptibility genes across the genome involved
in the disorder other than confirming a role of HLA.