2008 Barcelona - European Society of Human Genetics

Cancer genetics
of UVs newly found in patients .
We used different publicly available programs and web-based tools to
identify UVs that may have deleterious effects with respect to different
biomolecular functional categories (splicing regulation, transcriptional
regulation, nonsynonymous amino acid SNP effect . . .) so their clinical
significance in cancer etiology could be assumed.
Using straightforward physical and comparative considerations, we
have found that several sequence variants with nonsynonymous amino
acid change could have possible impact on the structure and function
of a BRCA1 and BRCA2 proteins . Synonymous amino acid changes
(silent mutations) could have impact on splicing regulation by disrupting
exonic splice enhancers . Intronic sequence variants showed no
potential impact on splicing because nucleotide changes at that positions
likely make no changes in consensus splice sites .
In Silico analysis of BRCA1 and BRCA2 presents fast, easy and cheap
method for assessing preliminary clinical significance for UVs, especially
in cases with low frequency and ethnic specific alleles, when
it is difficult to make population based studies and when expensive
experimental functional assays must be performed .
P04.069
three years of real-time PcR-based gene dosage for
BRCA large rearrangements in France : new perspective in
combination with HRm curve analysis
C. Lefol, E. Rouleau, G. Christophe, F. Copigny, C. Andrieu, I. Bieche, R. Lidereau;
Centre Rene Huguenin, St Cloud, France.
BRCA1 germline mutations are responsible for susceptibility in breastovarian
cancers . Some alterations are large rearrangements (15%) .
To detect them, our laboratory has proposed a real-time PCR-based
gene dosage . Since 2004, samples from all of over France have been
analyzed with this method to confirm large rearrangements detected
with semi-quantitative multiplex PCR (MLPA or QMPSF) . Recently, our
qPCR experience have been transfer to LightCycler 480 combining
High-Resolution Melting curve analysis .
For each genomic DNAs sent, suspected exons were scanned with
a few others . Copy numbers of each exon were calculated with an
algorithm based on delta Ct method . As reference of the diploidy, three
genes (4q, 8q, 17q) were quantified.
148 samples have been analysed with ABI Prism 7900 and PowerSybr
Mix. 49 rearrangements were confirmed (table). 99 samples have no
rearrangement . Most of those BRCA1 large rearrangements were analysed
and validated with LightCycler 480 and HRM Master Mix using
the same qPCR primers . Furthermore, by applying the same algorithm
and PCR conditions, selected HRM primers were also able to detect
those rearrangements .
This large series confirms the abilities of qPCR to detect large rearrangements
in a routine screening . HRM curve analysis combined with
quantitative PCR can give in unique assay information for point mutations
and large rearrangements . Therefore, this technique should be
reconsidered as a first-line tool for BRCA1 screening .
Result of 148 suspected BRCA1 large rearrangements by real-time
PCR-based gene dosage
Number of Number of con-
%
samples firmation
Deletion suspected 101 35 35%
Duplication suspected
16 4 25%
(5’region to exon 1-2)
Duplication suspected
31 10 30%
(others exons)
P04.070
characterization of some acute lymphoblastic leukemia cases
by cytogenetic technique
D. Usurelu1 , L. Gavrila1 , D. Cimponeriu1 , P. Apostol1 , L. Dan1 , I. Radu1 , V. Teleanu2
, A. Moicean2 , A. Dumitrescu2 ;
1 2 Institute of Genetics, Bucharest, Romania, Fundeni Hospital, Bucharest, Romania.
Cytogenetical analyses are important for diagnostics, prognostic and
monitoring of the treatment efficiency in leukemia.
Objective:
Cytogenetical characterization of some Romanian patients with acute
lymphoblastic leukemia (ALL) .
material and methods:
Five patients with ALL from Fundeni hospital, Bucharest were analyzed
.
Bone marrow cells were cultivated in specific medium for 96 hours. The
slides were prepared according to the classical procedure and stained
with Giemsa solution . 100 metaphases/patient were analyzed .
The cytogenetic analyses follow two directions:
- detection of chromosomal aberrations and identification of t(9;22)
- evaluation of a potential link between leukemic phenotype and the
telomeres structure .
Results and conclusion:
An obvious genomic instability of the leukemic cells was noticed, expressed
by different chromosomal rearrangements, as well as by genomic
mutation i .e . aneuploidy (table) .
Chromosomal rearrangements
Normal
Telomere-to-telomere
Patients
Aneuploidy
ring chromo-
metaphases PCD
and telomere-to- centro- Fusions
(>50chromosomes)
somesmere
attractions
1 43 10 1 8 12 0
2 55 15 3 2 7 1
3 29 11 0 1 5 0
4 48 13 8 3 3 2
5 35 20 5 7 7 0
The FISH analyses showed the t(9;22) was present in two patients. This translocation
was present in 57% of the metaphases of the first patient and in 71% of the metaphases
of the third one.
No correlation between the telomere length and the leukemic phenotype could be
detected. The fluorescence signal was encountered in more than 97% of the chromosomes,
that indicates almost intact telomeric repetition, without large terminal deletions.
The incidence of t(9;22) found in two out of five patients is comparable to other studies
taking in acount also that they didn’t receive any treatment, being at the diagnostic
stage.
P04.071
chromosomes 3 and 7 rearrangements in complex
chromosomal aberrations in patients with myelodysplastic
syndromes and acute myeloid leukemia
J. Brezinova 1 , Z. Zemanova 2 , L. Babicka 2 , S. Izakova 1 , J. Cermak 1 , M. Siskova
3 , K. Michalova 1,2 ;
1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic,
2 Center of Oncocytogenetics Institute of Clinical Biochemistry and Laboratory
Diagnostics General Faculty Hospital and 1st Faculty of Medicine of Charles
University, Prague, Czech Republic, 3 1st Medical Department General Faculty
Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech
Republic.
Rearrangements of chromosomes 3 and 7 are frequent in myelodysplastic
syndromes (MDS) and acute myeloid leukemia (AML) and are
associated with poor outcome and resistance to treatment . Combinations
of molecular cytogenetic techniques were used for identification
of these rearrangements in bone marrow cells of patients with complex
chromosomal aberrations (CCA) . In cohort of 392 patients with AML
and RAEBt (refractory anemia with excess blast in transformation) we
proved CCA in 58 of them . Chromosome 3 was involved in complex
aberrations in 26 patients, chromosome 7 in 23 patients, both chromosomes
3 and 7 in nine patients . The most frequent aberrations of
chromosome 3 were non-reciprocal translocation combined with deletion
of chromosome 3 (14 patients) . Other abnormalities proved by
FISH: non-reciprocal translocations without deletion (8 patients), reciprocal
translocation (5 patients), partial trisomy (4 patients), inversion
(1 patient) and insertion of a part of chromosome 3 into chromosome
2 (1 patient) . Breakpoints 3p24 .2 and 3q26 were the most frequent .
Minimal deleted segment 3p25-3pter was ascertained in 12 patients .
Similarly, large heterogeneity of the breakpoints and various extent of
deletion were proved on chromosome 7 with the most frequent breakpoints
7q31 and 7p12 . Partners in translocations were chromosomes
5, 3 and 12 . Using combination of molecular cytogenetic techniques
we found a wide variety of aberrations not detectable by conventional
cytogenetic analysis . We presume contribution of more genes and participation
of epigenetic factors in the origin and during the course of
the disease .
Supported by grants IGA NR 9227-3, MZO 00023736, MSMT LC535 .
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