12th Congress of the European Hematology ... - Haematologica

Cytogenetics and Molecular diagnostics I
0129
PARENTAL ORIGIN DETERMINATION I FISH A NEW METHOD TO CHARACTERIZE THE
PARENTAL ORIGIN OF HUMAN CHROMOSOMES ON A SINGLE CELL LEVEL
M. Gross, 1 H. Mkrtchyan, 2 H. Thieme, 1 F. Von Eggeling, 1
B. Horsthemke, 1 Chr. Jonsrud, 3 U. Claussen, 1 TH Liehr, 1 A. Weise1 1 Institute of Human Genetics, JENA, Germany; 2 Yerevan State University,
YEREVAN, Armenia; 3 Dept. of Medical Genetics, TROMSO, Norway
The discrimination of homologues (chromosomal) regions could up to
now be done exclusively by molecular genetic Aims.using microsatellite
or SNP-analysis. Only in exceptional cases a distinction on a single cell
level by chromosomes was possible. Therefore, we developed a method
called parental origin determination fluorescence in situ hybridization
(pod-FISH) that is suited for a single cell analysis and can distinguish
every homologues chromosome pair on the basis of copy number polymorphisms
(CNP). Presently we selected 225 of over 2191 reported polymorphic
regions in the human genome and used them as pod-FISH
probes. pod-FISH results were verified by microsatellite analysis. We
established pod-FISH probe sets for all 24 human chromosomes. To
enable working with more than one polymorphic BAC probe at the
same time, chromosome specific pod-FISH sets using 5 different fluorochromes
were developed. For larger chromosomes like chromosomes
1, 2, 3, 4, 6, 9 and X it was more convenient to create chromosome arm
specific pod-FISH sets for an easier analysis and to prevent double labeling.
Up to now two cases with a known UPD, 2 cases with maternal contamination
and three leukemia cases (AML with trisomy 8) were analyzed
successfully. Also the method was applied on a unique case which
is a chimera of two cell lines: one female cell line with a complete paternal
isodisomy (97%) and one normal male cell line (3%). The results
obtained by pod-FISH were in complete concordance with data obtained
by microsatellite-analysis. The usefulness and feasibility of the new pod-
FISH approach was compared to conventional microsatellite analysis
and proved to be as reliable. Thus, the pod-FISH method will open new
horizons for diagnostic and scientific fields that could not be questioned
by now. E.g. the analysis of single cells will allow in leukaemia to get new
diagnostic markers for therapy control after transplantations or to detect
maternal contamination in prenatal diagnosis.
Supported in parts by a grant from the university of Jena, the Deutsche Krebshilfe
(70-3125-Li1), the INTAS (AISbl 03-51-4060), the IZKF Jena (Start-up
S16), the DFG (436 ARM 17/5/06, LI 820/9-1), the IZKF together with the
TMWFK (TP 3.7 and B307-04004), Stiftung Leukämie and the Ernst-Abbe-
Stiftung.
0130
ALTERATIONS OF THE TP53 IN AML AND CLL ARE STRONGLY ASSOCIATED WITH
A COMPLEX ABERRANT KARYOTYPE
C. Haferlach, F. Dicker, H. Herholz, S. Schnittger, W. Kern,
T. Haferlach
MLL - Munich Leukemia Laboratory, MUNICH, Germany
The TP53 gene is the most frequently mutated gene in human tumors
identified so far. However, in leukemia TP53 alterations seem to be rare.
It was the aim of this study to determine the incidence of TP53 mutations
in AML and CLL and analyze their relation to cytogenetic and other molecular
genetic aberrations. Overall 235 AML cases were analyzed for TP53mutations,
FLT3-length mutations (FLT3-LM), MLL partial tandem duplication
(MLL-PTD) and NPM1 mutations. TP53 mutation screening of
exons 3-9 was performed by denaturing high performance liquid chromatography
(DHPLC). All mutations detected were verified by direct
sequencing. The first 149 patients were unselected, the remaining were
selected to increase the numbers in rare cytogenetic subgroups. In the unselected
cohort a TP53 mutation was detected in 13.4% (20/149 cases). In
the total cohort a TP53 mutation was observed in 33 of 235 cases (14%).
29 of these 33 cases (87.9%) showed a complex aberrant karyotype. In the
four remaining cases with TP53 mutation a normal karyotype, a trisomy
8, a trisomy 13, and a t(8;21)(q22;q22) each as the sole abnormality were
observed, respectively. This data confirms results from our previous independent
series demonstrating a high incidence of TP53 mutations in AML
with complex aberrant karyotype (1. series 78%, current series 69%). In
all other cytogenetic subgroups TP53 mutations are very rare (4/193,
2.1%). Two cases withTP53 mutation showed also a FLT3-LM and one a
MLL-PTD. TP53-mutations were not found together with NPM1 muta-
12 th Congress of the European Hematology Association
tions. In addition 194 consecutive CLL patients were screened for TP53deletion
by fluorescence in situ hybridization (FISH) and for TP53-mutations.
A TP53 deletion was detected by FISH in 18 cases (9.3%). 16 of these
18 cases also showed a TP53 mutation. In four further cases without a
TP53 deletion a TP53 mutation was observed. Therefore, the total incidence
of TP53 alterations in CLL was 11.3% (22/194) with a significant
association between TP53 deletions and TP53 mutations (p