2008 Barcelona - European Society of Human Genetics

Cancer genetics
P04.042
BRCA1/2 mutations: screening strategy for the analysis of
Portuguese high-risk breast/ovarian cancer families
M. Santos 1 , P. Machado 1 , S. Fragoso 1 , P. Rodrigues 2 , S. André 3 , F. Vaz 4 ;
1 Molecular Biology Departament-Portuguese Institute of Oncology, Lisbon,
Portugal, 2 Breast Cancer Risk Evaluation Clinic-Portuguese Institute of Oncology,
Lisbon, Portugal, 3 Pathology Department-Portuguese Institute of Oncology,
Lisbon, Portugal, 4 Molecular Biology Departament and Breast Cancer Risk
Evalution Clinic-Portuguese Institute of Oncology, Lisbon, Portugal.
Background: Five to ten percent of breast cancers are hereditary and
50-80% due to BRCA1/2 mutations . Because screening of BRCA1/2
mutations is complex and indeterminate results are frequent, only
high-risk patients should be counselled for this diagnosis . The possibility
of an informative result also depends on diagnostic methodologies
performed . Purpose: elaboration of an algorithm for BRCA1/2
mutation screening in Portuguese high-risk breast cancer families .
Methods: Integration of clinical and previous molecular data showing
that c .156_157insAlu in the BRCA2 gene is a Portuguese founder mutation
, BRCA1 immunohistochemistry helps in selection for screening
as well as the prevalence of multiplex ligation dependent probe amplification
(MLPA) detected rearrangements . Results: We suggest the
following algorithm: 1-All samples are first screened for the founder
mutation (6-8% positives); 2- if negative in 1 recurrent rearrangements
(g .Ex13ins6kb and g .Ex11_15del) are screened by PCR; 3- negatives
in 1 and 2 are screened by conformation sensitive gel electrophoresis .
The first gene to analyze depends on tumor phenotype: BRCA1 first
if BRCA1 immunohistochemistry is negative and/or is a triple negative
tumor. BRCA2 first if a BRCA1 phenotype is excluded and/or history
of male breast cancer or BRCA2 associated tumours (gastric, M .
mieloma, pancreas); 4- if still negative MLPA is performed .
Conclusion: This algorithm might fit into a complete screening program
and be superior to current practice in terms of providing more informative
results .
P04.043
correlation between BRcA1 mutations and BRcA1 expression
level in breast cancer specimens
N. Lojo - Kadric 1 , L. Kapur 1 , J. Ramic 1 , D. Macic 1 , N. Bilalovic 2 , K. Bajrovic 1 ;
1 Institute for genetic engineering and biotechnology, Sarajevo, Bosnia and Herzegovina,
2 Institute of Patology, KCUS, Sarajevo, Bosnia and Herzegovina.
Tumor suppressor BRCA1 gene is associated with increased risk for
developing breast and ovarian cancer . Approximately 80% of hereditary
breast cancer cases have mutations in BRCA1 gene . This gene
is expressed in several organs, including breast and ovary . BRCA1
encodes a 220 kDa protein which consist of 1863 amino acids . Expression
of BRCA1 gene is decreased in the most early - detected
sporadic breast cancer cases, and it decreases continuously with degree
of malignancy .
The goal of this experiment was to investigate whether expression
of BRCA1 is affected by mutations on BRCA1 gene . Tumor bioptic
specimens from patients fulfilling criteria to belong to high to medium
risk group for breast cancer development have been consecutively collected
at Sarajevo Clinical Center during the period of two years . DNA
was isolated from tumor tissue samples using standard salting out procedure.
Mutations in BRCA1 gene were identified by MLPA reaction.
For expression level measurement, RNA from tumor specimens was
isolated using guanidine isotiocyanate method, followed by reverse
transcription . Measurement of expression levels of BRCA1 gene was
performed in 7300 Real Time PCR system (Applied Biosystems) with
SYBR green method . After collecting data, correlation calculations
were performed . Results from MLPA analysis were correlated with
expression levels of BRCA1 gene . Results are displayed in graphic
figures.
P04.044
the 3-step PcR methodology is the correct option to screen the
c. _ insAlu BRCA Portuguese founder mutation
P. M. Machado 1 , A. Hardouin 2 , S. Santos 1 , S. Fragoso 1 , P. Rodrigues 3 , S.
Bento 3 , S. Braga 3 , A. Luís 4 , A. Esteves 3 , F. Vaz 5 ;
1 Molecular Biology Department-Portuguese Institute of Oncology, Lisbon,
Portugal, 2 Laboratoire de Biologie Clinique et Oncologique- Centre François
Baclesse, Caen, France, 3 Breast Cancer Risk Evaluation Clinic-Portuguese
Institute of Oncology, Lisbon, Portugal, 4 Breast Cancer Risk Evaluation Clinic
- Portuguese Institute of Oncology, Lisbon, Portugal, 5 Molecular Biology Department
and Breast Cancer Risk Evaluation Clinic-Portuguese Institute of Oncology,
Lisbon, Portugal.
introduction: The insertion of an Alu fragment in position c .156_157
of BRCA2, causing exon 3 skipping, was confirmed to be a Portuguese
founder mutation and the most frequent BRCA2 rearrangement
described . We also optimized a methodology for the screening of this
mutation . Patients and methods: All new high-risk patients counselled
for BRCA1/2 screening in our Centre are pre-screened for this
mutation with a 3-step PCR method (Patent pending): 1) exon 3 PCR
and sequencing, 2) nested PCR with primers 3AluF/3AluR and 3) RT-
PCR and cDNA sequencing . Other Centres, in Portugal and abroad,
are implementing this PCR-based method to screen for this mutation .
Additional family inquiries are made due to the founder effect of this
mutation . Results: Twenty-nine apparently nonrelated Portuguese
c.156_157insAlu-positive families were diagnosed in our Centre and
one BC/OC family of Portuguese ancestry was also diagnosed in
Caen, France, using our method. Also, a first degree relative of one
of our new probands had been studied in USA and diagnosed with an
unknown variant (Q2731E) . Exhaustive family inquiries allowed us to
connect 3 of these new and apparently non-related families with families
already identified. conclusion: The correct option to screen the
c.156_157insAlu BRCA2 Portuguese founder mutation is the 3-step
PCR method we described . Our data suggests that other methodologies
may miss this mutation, preventing these families from obtaining
an informative result .
P04.045
Prevalence of large rearrangements in Portuguese hereditary
breast/ovarian cancer families
S. Fragoso 1 , J. Eugénio 1 , P. Machado 1 , S. Santos 1 , P. Rodrigues 2 , H. Vicente 2 ,
S. Bento 2 , A. Esteves 2 , F. Vaz 3 ;
1 Molecular Biology Department- Portuguese Institute of Oncology, Lisbon,
Portugal, 2 Breast Cancer Risk Evaluation Clinic- Portuguese Institute of Oncology,
Lisbon, Portugal, 3 Molecular Biology Department and Breast Cancer Risk
Evaluation Clinic- Portuguese Institute of Oncology, Lisbon, Portugal.
Background: Hereditary breast/ ovarian (HBO) cancer is mainly related
with BRCA1/2 mutations . Given the increasing number of rearrangements
reported and the observation that the most frequent BRCA
mutation in our country is a BRCA2 rearrangement (c .156_157insAlu),
present in about 50% of our BRCA1/2 positive families, the contribution
of other large genomic rearrangements to the Portuguese BRCA
mutation spectrum must be analysed . Aim: to evaluate the frequency
of large rearrangements in the BRCA1/2 genes in Portuguese HBO
families . Patients and methods: Ninety high-risk families, previously
negative for BRCA1/2 mutations by conformation sensitive gel electrophoresis,
were screened for the g .Ex13ins6Kb BRCA1 by PCR using
specific primers and multiplex ligation-dependent probe amplification
(MLPA) . Results: Two additional rearrangements, both in the BRCA1
gene, were observed: g .Ex13ins6Kb (1 family) and g .Ex11_15del (1
family), accounting for 22% (2/9) of the BRCA1 mutation spectrum in
our families . With the MLPA BRCA2 kit, 1 patient was found to be
positive for the 1100delC mutation in CHEK2 and 3 patients had copy
number changes in this gene . No large BRCA2 rearrangements were
observed . conclusion: Besides the screening of the founder BRCA2
rearrangement in our population, index high-risk cases negative for
BRCA1/2 point mutations should be analysed for other possible genomic
BRCA rearrangements . Although infrequently, CHEK2*1100delC
is present as a low penetrance allele in our population . Additional studies
are necessary to clarify the relevance of copy number changes in
CHEK2 gene .
P04.046
Zoom-in Array-CGH in BRCA , BRCA , MLH , MSH and APC
genes : detection and characterization of five new germline large
rearrangements
E. Rouleau1 , S. Tozlu1 , C. Andrieu1 , C. Lefol1 , V. Bourdon2 , T. Nogushi2 , C.
Nogues1 , S. Olschwang2 , H. Sobol2 , I. Bieche1 , R. Lidereau1 ;
1 2 Centre Rene Huguenin, St Cloud, France, Institut Paoli-Calmettes, Marseille,
France.
Genetic predisposition to breast, ovarian, colorectal cancers results
mainly from alterations in BRCA1, BRCA2, MLH1, MSH2 and APC
genes . Except for BRCA2, rearrangements represent 10 to 15% of
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