2008 Barcelona - European Society of Human Genetics

Cancer genetics
Method: mRNA level of MRP1 was determined in 111 patients with
acute leukemia including 52 patients with AML and 59 patients with
ALL by RT-PCR and compared to the type of response to chemotherapy
.
We found that overexpression of MRP1 is indeed and associated with
MDR phenotype in leukemic patients .
Furthermore, the128G>C , 816G>A , 825T>C,1299G>C and -
260G>C,-275
A>C were genotyped in all the patients and control group .
P04.099
PHLPP gene is mutated in pediatric acute myeloid leukemia
patients and might act as a tumor suppressor gene
T. A. Tekiner 1,2 , F. Atalar 1 , A. Karabay-Korkmaz 2 , S. Anak 3 , U. Ozbek 1 ;
1 Istanbul University, Institute of Experimental Medical Research (DETAE), Genetics
Department, Istanbul, Turkey, 2 Istanbul Technical University, Molecular
Biology and Genetics Department, Istanbul, Turkey, 3 Istanbul University, Istanbul
School of Medicine, Department of Pediatric Hematology and Oncology,
Istanbul, Turkey.
The overactivated PI3K/Akt pathway represents potential therapeutic
targets for AML . The protein phosphatase PHLPP has been shown recently
to specifically dephosphorylate S473 of Akt which regulates the
balance between cell survival and apoptosis . Human PHLPP contains
an amino-terminal PH domain, a leucine-rich repeat region (LRR), a
PP2C-like catalytic core and a PDZ binding motif . So far, there are
no described mutations in PHLPP gene . In this study, we aimed to
understand the architecture of PHLPP gene variations in pediatric AML
patients . We report here the molecular screening results of 11 exons
covering the four domains of PHLPP gene in 38 pediatric AML patients
. The screening for the presence of a mutation was performed
by dHPLC analysis . Mutation detection was accomplished by direct
sequencing .
We found the following sequence variations, exon2 and 3; 59insA(5 .2
%),60C>T(2 .8%), 77C>A(2 .8%),109A>T, 289C>A(7 .8%),352C>A(7 .8
%),343insA(2 .8%), exon5, 6 and 7; 599insA(47%), exon14,15,16,17
and 18; 1980T>C(5 .2%),1992T>C(5 .2%), exon19; 3280C>A(13 .1%
),3302insA(13,1%),3303T>C(13 .1), 3407insA(5 .2%) and 3611insC
(7 .8%) . The expression analysis of the exons covering the four domains
were performed by QRT-PCR . Interestingly, in all patients, we
could not detect any expression in LRR domain . We are currently investigating
the effect of 599insA (S200R), which might result structural
and functional changes at protein level due to the change of the amino
acid charges. This is the first study evaluating sequence variations together
with the expression of PHLPP gene . We propose that PHLPP
gene might act as a tumor suppressor in AML leukomogenesis and this
can provide an important guidepost for the development of diagnostic
tools for acute leukemia .
P04.100
Beta catenin gene expression and mutation analysis in t-ALL
patients and cell line
Y. Erbilgin, M. Aydin Sayitoglu, O. Hatirnaz, U. Ozbek;
Institute of Experimental Medical Research, Istanbul, Turkey.
The molecular mechanisms regulating the development and differentiation
of the haematopoietic system are not clearly understood . Recent
studies showed that Wnt signaling proteins play significant roles
in both normal lymphocyte development and leukemia pathogenesis .
Wnt proteins activate a complex signaling cascade, leading stabilization
of β-catenin, which is a key component of the Wnt signaling
pathway. β-catenin levels are regulated post-translationally by the canonical
Wnt signaling pathway. In this study, we investigate the Wnt/βcatenin
pathway activation in T- cell acute lymphoblastic leukaemia
(T-ALL) patients and cell lines .
We analyze β-catenin mRNA expression using quantitative real time
PCR (QRT-PCR) . We measured protein levels by western blot and
search for mutations in exon 2-3 of the β-catenin gene in 73 T-ALL
patients that were applied to our department for molecular diagnostic
purposes and 29 T-ALL cell lines . Wnt signals are transduced by active
β-catenin. We showed that T-ALL patients and cell lines have abnormal
nuclear accumulation of β-catenin. We also found a β-catenin
gene (exon3) mutation in one T-ALL patient . To the best of our knowledge,
this is the first study that shows β-catenin gene mutation in T-
ALL patients. No mutation was found in any of the cell lines. Our find-
ings reconfirm that Wnt signaling is deregulated in T-ALL by abnormal
β-catenin expression. We discuss that β-catenin may play a significant
role in promoting leukemic cell proliferation, adhesion, and survival via
increased transcription of Wnt target genes .
P04.101
Deregulation of IRS as a result of juxtaposition to t-cell
receptor beta in a pediatric t(X;7)(q22;q34)-positive t-cell acute
lymphoblastic leukemia
K. Karrman1 , C. Lassen1 , E. Kjeldsen2 , T. Fioretos1 , B. Johansson1 ;
1 2 Department of Clinical Genetics, Lund, Sweden, Cancer Cytogenetic Laboratory,
Aarhus, Denmark.
OBJECTIVES: A t(X;7)(q22;q34), an abnormality not previously described
in hematologic malignancies, has been molecularly characterized
in a pediatric T-cell acute lymphoblastic leukemia (T-ALL) . PA-
TIENTS AND METHODS: The t(X;7), initially detected by G-banding
analysis, was further investigated with fluorescence in situ hybridization
(FISH), real-time polymerase chain reaction (PCR) and Western
blot analysis . RESULTS: FISH disclosed a breakpoint in the T-cell receptor
beta locus (7q34) and a breakpoint between RP11-815E21 and
RP11-105F23 mapping at Xq22 .3 . The two genes located closest to
this region, i .e . insulin receptor substrate 4 (IRS4) and collagen, type
IV, alpha 5 (COL4A5), were analyzed using real-time PCR . COL4A5
was not differentially expressed in the t(X;7)-positive sample compared
to five T-ALL controls. However, a marked overexpression of IRS4 was
identified in the (X;7)-positive case in relation to the controls. Although
the Western blot analysis was suboptimal due to protein degradation,
a band representing IRS4 was found in the t(X;7)-positive T-ALL; this
band was not seen in the control samples . CONCLUSION: We report
the first T-cell neoplasm with a translocation resulting in deregulation
of IRS4 . In fact, this is so far the only reported neoplasia in which a
member of the IRS family has been implicated .
P04.102
Clinical significance and prevalence of T315I mutation in Indian
cmL patients treated with imatinib mesylate
R. Mir, S. Sazawal, R. Chobey, S. Bharti, A. Chopra, P. Mishra, B. Bohra, R.
Saxena;
Department of Haematology, All India Institute of Medical Sciences, New Dehli,
India.
Background: The early detection of T315I mutations may allow timely
treatment intervention to prevent or overcome resistance .
Lacunae: Prevalence of T315I mutation in Indian CML patients .
Aims: To detect T315I mutation in CML patients by ASO-PCR . To study
prevalence of T315I mutation in Indian CML patients .
Methodology: CML patients were diagnosed by RT-PCR .ASO-PCR
was done for all 160 patients for BCR-ABL mutations especially for
T315I . The patients were evaluated for hematologic and molecular responses,
time to progression, survival and toxicity .
Results : The study included 160 CML patients . The mutation was detected
in 30% of patients (30/160) . The median time of Gleevec treatment
25 months .The onset of T315I mutation in 30 patients developed
poor prognostic factors in these patients . 25/30 lost hematological &
molecular responses .20/25 progressed to advanced stage . T315I mutations
had proven to be fatal & is soul cause of Imatinib resistance in
our patients . Survival and time-to-progression curves were obtained
from Kaplan-Meier method .
Discussion : India is a developing country, the patients cannot effort
expensive tests like sequencing for T315I mutation . So we had standardized
ASO-PCR for routine screening of T315I mutations in our
patients .
Conclusion: ASO-PCR proved to be very economical, sensitive and
rapid technique for detection of known T315I mutations and is even
sensitive than mutation detection by sequencing . The early detection
by ASO-PCR assay proved to be helpful in clinical management of
therapeutic decisions in CML patients .