2009 Vienna - European Society of Human Genetics

Cancer genetics
BRCA1 and BRCA2 account for 20% of familial breast cancers. For
the remaining 80%, genetic factors are largely unknown. Recently, a
new BRCA1-interacting protein, RAP80, was identified. RAP80 plays
an important role in BRCA1-mediated DNA damage responses by recruiting
BRCA1 to DNA double-strand breaks (DSB).
Objective: To investigate whether germline mutations in RAP80 are
associated with an increased risk of developing cancer.
Methods: Mutation screening was performed by CSGE and direct sequencing.
For one of the observed alterations also functional studies
were performed to assess influence on 1) Ubiquitin binding 2) RAP80
DSB localization 3) RAP80-BRCA1 complex DSB localization and 4)
Genomic instability.
Results: Mutation screening from 112 index cases of Finnish breast
cancer families revealed 10 alterations in RAP80, one of which was a
novel exonic change. This novel alteration resulted in a dysfunctional
protein product that displayed significantly reduced ubiquitin binding
and double-strand break localization after DNA damage. It also impaired
both BRCA1 and ABRA1 double-strand break recruitment, thus
compromising BRCA1-mediated DNA damage response signaling. A
significant increase in cytogenetically detectable chromosomal aberrations,
particularly chromatid breaks, was also observed.
Conclusions: These results suggest that germline mutations in RAP80
abrogate DNA damage response function and may be involved in genetic
predisposition to cancer.
P06.081
Aberrant methylation of the genes VHL, RASSF , FHIT, SFRP ,
and CDH in clear cell renal cancer
D. S. Mikhaylenko 1,2 , A. M. Popov 3 , R. V. Kurynin 4 , L. E. Zavalishina 5 , D. V.
Zaletayev 1,2 ;
1 Research Centre for Medical Genetics RAMS, Moscow, Russian Federation,
2 Institute of Molecular Medicine of Sechenov Moscow Medical Academy,
Moscow, Russian Federation, 3 Medical Radiological Research Center RAMS,
Obninsk, Russian Federation, 4 Clinic of Urology of Sechenov Moscow Medical
Academy, Moscow, Russian Federation, 5 Hertzen Oncological Research Institute,
Moscow, Russian Federation.
Clear cell renal cancer (CCRC) is the most common tumor of the kidney,
200 thousands CCRC cases are registered worldwide annually.
CCRC is characterized by tumor suppressor genes inactivation owing
to several mechanisms including methylation. We have conducted the
study of methylation of the genes VHL, RASSF1, FHIT, SFRP1, and
CDH1 in 123 CCRC for the development of renal cancer diagnostic
and prognostic criteria. Methylation was detected by methylsensitive
endonuclease BstHHI digesting and following PCR, hypermethylated
samples were confirmed using bisulphite sequencing. Aberrant methylation
of VHL was observed in 14.6% (18/123), RASSF1 - 53.7%
(66/123), FHIT - 54.5% (67/123), SFRP1 - 34.1% (42/123), and CDH1
- 43.1% (53/123) cases. Methylation of at least one gene from these
was detected in 85.4% (105/123) samples. CDH1 methylation was associated
with tumor invasion through the kidney capsule (P = 0.024)
and metastases in the regional lymph nodes and/or distant metastases
(P = 0.001). It was found that RASSF1 was methylated more
frequently in primary tumors with grade G 2 than G 1 (P = 0.047). The
genes studied could be used with some others tumor suppressors for
formation of a diagnostic panel containing the genes frequently methylated
in renal cancer, and aberrant methylation of the genes CDH1
and RASSF1 could indicate a primary tumor progression on different
stages of CCRC.
P06.082
Identification of transcriptional targets by ChIP-Sequencing in
t(X;1)-positive renal cell carcinomas
L. Brugmans, L. Hetterschijt, L. Vreede, K. Medendorp, A. Geurts van Kessel;
Radboud university Nijmegen MC, Nijmegen, The Netherlands.
Previously, we and others showed that in a subset of human renal cell
carcinomas the bHLH-LZ transcription factor TFE3 is recurrently fused
to a novel protein designated PRCC. Subsequently, we established
that the resulting PRCCTFE3 fusion product acts as an oncogenic protein,
both in vitro and in vivo. In addition, we found that PRCCTFE3
acts as a more potent transcriptional activator than wild-type TFE3.
More recently, a functional cDNA screen revealed that TFE3 over-expression
renders cells insensitive to the anti-proliferative effects of the
G1/S cell cycle regulator pRB. We propose that also the PRCCTFE3
fusion protein may act through a cell cycle-mediated deregulation of
proliferation. In order to identify downstream transcriptional targets of
the PRCCTFE3 fusion protein, we initiated to use chromatin immunoprecipitation
(ChIP). Specifically, we are employing a recently developed
variant of this technology, called ChIP-Sequencing, which combines
ChIP with massive parallel sequencing to identify and quantify in
vivo protein-DNA interactions on a genome-wide scale. The identification
of novel PRCCTFE3 transcriptional targets and its implications
for our understanding of the role of cell cycle (de-) regulation in renal
tumor development will be discussed.
P06.083
the tyrosine kinase REt interacts in vivo and in vitro with AiP
M. Vargiolu 1 , D. Fusco 1 , I. Kurelac 1 , L. F. Pennisi 1 , E. Mariani 1 , M. Vidone 1 , D.
Dirnberger 2 , R. Baumeister 2 , I. Morra 3 , A. Melcarne 4 , R. Rimondini 5 , G. Romeo 1 ,
E. Bonora 1 ;
1 U.O. Genetica Medica, Policlinico S. Orsola-Malpighi via Massarenti 9, Bologna,
Italy, 2 Bio3/Bioinformatics and Molecular Genetics (Faculty of Biology),
University of Freiburg, Germany, 3 Department of Histopathology, Ospedale
Infantile Regina Margherita, Torino, Italy, 4 Department of Neurosurgery, A.S.O.
CTO-CRF-M.Adelaide, Torino, Italy, 5 Dept. of Pharmacology, University of Bologna,
Via Irnerio 48 Bologna, Italy.
RET is a tyrosine kinase transmembrane receptor expressed in two
main alternative isoforms: RET9 and RET51. RET transduces a
positive signal leading to survival, differentiation or migration in the
presence of its ligand GDNF, whilst in absence of the ligand, RET is
cleaved generating a proapoptotic fragment which initiates a signalling
pathway for apoptosis. Hitherto, signal transduction leading to apoptosis
is still unclear.
We performed a screening to identify the interacting proteins of the
long isoform of RET, using a modified two-hybrid yeast complementation
assay, the split-ubiquitin system against a human brain expression
library.
One of the proteins we identified with this method was the aryl hydrocarbon
receptor interacting protein (AIP), a tumor-suppressor protein
recently found mutated in pituitary adenoma. We showed that RET-AIP
interaction was maintained both in cell lines of different origin (human
embryonic kidney and neuroblastoma) and in pituitary gland in vivo. In
addition, we identified the pro-apoptotic domain of RET as responsible
for AIP interaction, regardless of the presence of pituitary adenoma
specific mutations. AIP and RET genes were sequenced in 28 pituitary
adenoma but no relevant mutation has been found. Finally, we showed
that AIP-RET interaction does not require RET kinase activity or kinase
dependent signal transduction and it prevents the formation of AIPsurvivin
complex. The identification of AIP-RET complex represents
a starting point for studying key cellular processes involved in RET
induced apoptosis.
P06.084
ms-mLPA to study the contribution of epigenetic silencing in
Retinoblastoma.
M. Amenduni 1 , M. Mucciolo 1 , M. Bruttini 1 , K. Sampieri 1 , M. Mencarelli 1 , M.
Epistolato 2 , P. Toti 2 , A. Marozza 1 , F. Mari 1 , T. Hadjistilianou 3 , S. De Francesco 3 ,
A. Acquaviva 4 , F. Ariani 1 , A. Renieri 1 ;
1 Medical Genetics, Siena, Italy, 2 Department of Human Pathology and Oncology,
Siena, Italy, 3 Retinoblastoma Referral Center, Department of Ophtalmology,
Siena, Italy, 4 Department of Pediatrics, Obstetrics and Reproductive Medicine,
Italian retinoblastoma registry, Siena, Italy.
Recent studies in the field of DNA methylation have lead to the awareness
that epigenetic changes may represent an alternative or complementary
mechanism to mutational events in tumour progression. In
particular methylation of CpG islands in the promoter regions of a large
number of tumour suppressor genes is observed in several human
cancers. Previous studies on Retinoblastoma (RB) tissues showed
frequent hypermethylation of the DNA-repair genes MGMT and MLH1
and the tumor suppressor gene RASSF1A. Methylation-specific
MLPA (MS-MLPA) has been recently described as a method that allows
the simultaneous identification of epigenetic changes at multiple
sites. We applied this technique to study epigenetic changes in 10 RB
samples and we compared results to those obtained in normal retina.
Tumour tissues showed frequent hypermethylation of MGMT (70%),
MSH6 (60%), CD44 (50%), PAX5 (50%) and GATA5 (30%). Since
these genes are involved in DNA repair (MSH6), cellular differentia-