2008 Barcelona - European Society of Human Genetics

Cancer genetics
P04.033
Rapid screening of BRcA1 and BRcA2 spanish mutations by
High Resolution melting analysis
I. de Juan Jiménez, E. Esteban Cardeñosa, S. Palanca Suela, E. Barragan
González, P. Bolufer Gilabert;
Molecular Biology Laboratory, Valencia, Spain.
Background: BRCA1 and BRCA2 are the major genes responsible for
hereditary breast (BC) and/or ovarian cancer . Since BRCA1/2 are large
genes the majority of mutation detection procedures include screening
methods which are time-consuming and expensive . High-resolution
melting (HRM) is a recent promising screening method that combines
the simplicity, cost-effectiveness and rapid identification of genetic
variants . The aim of the study is to evaluate HRM in the screening of
the reported Spanish mutations .
Methods: We studied 40 BRCA1 and 47 BRCA2 DNA samples containing
different Spanish mutations . We also included a reference
group of 20 unknown DNAs from patients with sporadic BC . The assay
was carried out on LightCycler 480 (Roche) using LightCycler® 480
HRM Master and performing 21 and 25 independent PCR for the study
of BRCA1/2 gene mutations, respectively . The melting analysis was
performed with LightCycler® 480 Gene Scanning Software v .1 .0 . All
mutations and genetic variants detected were confirmed by sequencing
the PCR products .
Results: The HRM could discriminate the 87 BRCA1/2 Spanish mutations
analyzed from wild-type DNA . Besides, 82 out 87 mutations were
clearly differentiated from each other . In the sporadic BC group we did
not detect any deleterious mutations, however, we detected polymorphisms
and unclassified variants in both genes.
Conclusions: HRM is a valuable method for rapid screening of BRCA1/2
Spanish mutations . Besides, this procedure allows the genotyping of
different mutations included and is capable of differentiating unknown
genetic variants present in the PCR product .
Acknowledgements: to the “Fundación para Investigación La Fe”, FIS
PI060505 and AP-042/07 .
P04.034
Novel deleterious BRCA1/2 mutations found in the population of
Eastern spain
E. Esteban Cardeñosa1 , S. Palanca Suela1 , I. de Juan Jiménez1 , E. Barragán
González1 , I. Chirivella González2 , Á. Segura Huerta2 , C. Guillén Ponce2 , E.
Martínez de Dueñas2 , P. Bolufer Gilabert1 ;
1 2 Molecular Biology Laboratory, Valencia, Spain, Units of Genetic Conselling in
Cancer of the Valencian Community, Valencia, Spain.
From the identification of BRCA1 and BRCA2 as major genes responsible
of hereditary breast and/or ovarian cancer more than 1,800 pathogenic
mutations have been reported in the Breast Cancer Information
Core, many of them related with ethnic and/or geographic conditions
(Neuhausen SL, 1999) . The objective of the study is to describe the
twenty novel deleterious mutations identified in the Valencian Community
.
In the three first years of performance of the Program of Genetic Counselling
in Cancer of the Valencian Community we studied the BRCA1
and BRCA2 mutations in 596 unrelated breast/ovarian cancer patients.
The method followed consisted of multiplex PCR amplification
of all exons of the genes, mutation screening by the study of homoduplex/heteroduplex
and mutation identification by sequencing of the
PCR products .
We found 52 different deleterious mutations (21 in BRCA1 and 31 in
BRCA2), among the 112 positive index patients . In BRCA1 gene we
found 12 frameshift, 3 nonsense, 3 missense and 3 splicing mutations .
In BRCA2 we found 24 frameshift, 5 nonsense and 2 splicing . Twenty
of these mutations have not been reported previously, 5 in BRCA1
(c .1689delG, c .3700delC, p .Y1429X, c .4424_4425delCT and c .5430_
5452del23) and 15 in BRCA2 (c .489_490delCT, c .1835insT, c .1874_
1877delAGGA, c .2929delC, c .5025delT, c .5344_5347delAATA,
c .5946_5947dupCT, c .6118delA, c .6722delT, c .7400insA, p .Y2621X,
c .8270_8271delCA, c .8860+2T>G, c .9218delA and p .Q3156X) .
From our data two main conclusions could be drawn: (a) the remarkable
proportion of novel mutations (20/52; 38 .4%) and (b)that the majority
of these mutations (15/20; 75%) were found in BRCA2 gene .
P04.035
BRCA1 and BRCA2 variability in breast/ovarian cancer families
from the Basque country
I. -. Zubillaga1 , M. Carrera2 , K. Mugica2 , C. Vidales1 ;
1 2 Policlinica Gipuzkoa, San Sebastian, Spain, Instituto Oncologico, San Sebastian,
Spain.
BRCA1 and BRCA2 are tumor suppressor genes that are related with
inherited susceptibility to breast/ovarian cancer . We are analyzing the
polymorphic variants of BRCA1 and BRCA2 genes in families with clinical
criteria of hereditary breast/ovarian cancer . After DNA isolation, the
mutational analysis were performed by DNA sequencing of all BRCA1
and BRCA2 exons and flanking regions. Twentythree different types
of BRCA2 and twenty BRCA1 polymorphic mutations were identified
in twenty breast/ovarian cancer families . The patients selection was
made after a genetic counseling procedure, according the diagnostic
criteria recommended by de Spanish Society of Medical Oncology
(SEOM) and also using the BRCAPRO informatic model .
P04.036
Novel and common BRCA1 mutations in familial breast/ovarian
cancer patients from Lithuania
R. Janavicius 1,2 , J. Kasnauskiene 1,2 , V. Kucinskas 1,2 ;
1 Vilnius University Hospital Santariskiu Clinics, Medical Genetics Center, Vilnius,
Lithuania, 2 Department of Human and Medical Genetics,Vilnius University,
Vilnius, Lithuania.
Background: Mutations in the BRCA1 gene are found in many families
with multiple cases of breast and ovarian cancer . Twenty seven unrelated
female patients diagnosed with breast cancer and/or ovarian
cancer have attended cancer genetics clinic during period of 2006-
2007 year .
Methods: BRCA1 gene testing was initiated with direct sequencing for
common founder mutations: 4153delA in exon 11, 5382insC in exon
20 and enzyme restriction digestion for 300T/G in exon 5 . After the
exclusion of common mutations, multiplex ligation-dependent probe
amplification (MLPA) and prescreening by denaturing gradient gel
electrophoresis (DGGE) with confirmation by direct sequencing were
performed .
Results: Overall, pathogenic BRCA1 gene mutations were prevalent
in 8 (30%) unrelated proband patients: 3 (15%) with breast cancer
(two 5382insC, one 4153delA), 2 (50%) with ovarian cancer (5382insC
and 4153delA each) and 3 (100%) with breast and ovarian cancer
(5382insC, 4153delA and novel previously not reported deletion
4635delG in exon 15) . No 300T/G mutation and BRCA1 rearrangements
were detected .
For patients diagnosed with breast cancer ≤35 year, BRCA1 mutation
detection rate was 27% (3/11) and mutations were absent in breast
cancer patients group diagnosed after 36 years (0/6) .
Conclusions: Testing for two common BRCA1 mutations should be
considered in female cancer cases* when: (a) breast cancer diagnosed
before the age of 36 year; (b) medullary breast carcinoma; (c)
breast and ovarian cancer; (d) nonmucinous epithelial ovarian tumor
regardless the age (*or first degree relative/second through male). Full
BRCA1 gene analysis is warranted in patients with strong family history
of breast cancer .
P04.037
Occurrence of BRcA1 cryptic splice site mutation: iVs5-12A>G
in hereditary breast cancer families in the czech republic
E. Machackova1 , J. Hazova1 , S. Tavandzis2 , M. Lukesova1 , L. Foretova1 ;
1 2 Masaryk Memorial Cancer Institute, Brno, Czech Republic, JG Mendel Cancer
Centre, Novy Jicin, Czech Republic.
High Resolution Melting (HRM) analysis revealed presence of BRCA1
cryptic splice site mutation IVS5-12A>G in high-risk breast/ovarian
cancer patients . This change was described to form a cryptic splice
site resulting in the addition of 11 nucleotides at the intron 5/exon 6
boundary . The BRCA1 mutation IVS5-12A>G was not detectable by
the previously used heteroduplex analysis (using MDE gel) at the
MMCI . Therefore, the retrospective study of 480 high-risk familiar
breast/ovarian cancer patients without previously identified BRCA1/2
mutation were analysed for exon 6 by HRM using LightScanner instrument
. The BRCA1 mutation IVS5-12A>G was found in 11 high-risk
probands. Adjoining these new findings to our previous results, the
IVS5-12A>G mutation in BRCA1 gene represents 5,3% of all BRCA1