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

0438
TERC MUTATIONS ANALYSIS IN PATIENTS WITH APLASTIC ANEMIA: RESULTS OF A DHPLC
SCREENING AND CLINICAL IMPLICATIONS
S. Pigullo, 1 C. Dufour, 1 G. Caridi,1 J. Svahn, 1 G. Santamaria, 1
M. Risso, 1 M. Van Lint, 2 D. Longoni, 3 M. Pillon, 4 A.P. Iori, 5 P. Saracco, 6
A. Bacigalupo, 7 C. Di Grazia, 2 M. Lanciotti 1
1 G. Gaslini Hospital, GENOA; 2 San Martino Hospital, GENOA; 3 San Gerardo
Hospital, MONZA; 4 Padoa University, PADOA; 5 La Sapienza University,
ROME; 6 Regina Margherita Hospital, TURIN; 7 St. Martin Hospital,
GENOA, Italy
Background. Telomerase RNA Component (TERC) is an essential subunit
of the telomerase ribonucleoprotein complex. TERC mutation
results in a reduction of telomerase activity leading to premature telomere
shortening. Heterozygous mutations of TERC gene are responsible
for the autosomal dominant form of Dyskeratosis Congenita (DC).
Recently TERC mutations were also described in individuals with aplastic
anemia (AA) acquired apparently late in life. Denaturing High Performance
Liquid Chromatography (DHPLC) is a relatively novel technique
of mutation detection based on the separation of heteroduplex PCR
products from their corresponding homoduplex by reverse phase liquid
chromatography. Aims. To investigate the presence and the frequency of
TERC mutations in a population of Italian AA patients including pediatric
and adults subject. Methods. DNA from 111 AA patients (75 pediatrics
and 36 adults) and 156 normal controls (94 pediatrics and 62 adults
was obtained from blood or bone marrow samples previously collected
and frozen. The TERC coding region (GenBank NR_001566) was amplified
in 2 PCR fragments and then analyzed by DHPLC. For each abnormal
elution profile PCR products were directly sequenced using ABI
prism 3100 genetic analyzer. Results.Two new mutations, c53T>A and
c210C>G, of TERC gene were identified. The c53T>A mutation results
in a nucleotidic change in the template sequence inside the highly conserved
region CR1, but it does not alter TERC-RNA secondary structure.
This mutation was found in a 14 year old boy who, throughout his 10
year clinical follow-up, on CyA and Steroid to which he was not thoroughly
compliant, maintained hypocellular marrow and a mild cytopenia
(WBC 2.8-7.0×10 9 /L, PMN 1.0-4.5×10 9 /L, Hb 14-16 g/dL, Plt 60-80
×10 9 /L). The c210C>G mutation was found in a 47 year old woman and
localized in the P1 region. This mutation gives rise to a radical conformational
change of the TERC-RNA secondary structure. This patient
was diagnosed 10 years ago with severe AA, she did not respond to initial
treatment with ATG plus CyA. Afterwards, androgens and steroids
were added to CyA, she improved her counts (Platelets 50×10 9 /L, Hb 9.4
g/dL, PMN 3.0×10 9 /L), after 3 years of this therapy. In the last year, without
treatment, her Hb is 12 g/dL, Platelets 180×10 9 /L and PMN 3.0
×10 9 /L). Marrow committed progenitors are far lower than normal controls.
Conclusions. The frequency of TERC mutations in our cohort of AA
patients is low (1.75%) but consistent with previously published data.
We developed a fast, effective and low cost DHPLC-screening protocol
which allows TERC gene mutation analysis in all new cases of AA. Identification
of TERC mutations in AA patients has some important clinical
implications: i) the genetic test is useful for a correct diagnosis and
an adequate therapeutic protocol since TERC mutated patients often
have no other signs of DC and invariably fail to respond to immunosuppressive
therapy. ii) TERC mutation carriers, as they have a hereditary
disease with an elevated cancer-susceptibility, require an adequate cancer
surveillance program and genetic counseling.
0439
ADIPONECTIN IS PRODUCED BY LYMPHOCYTES AND INHIBITS GRANULOPOIESIS
L. Crawford, 1 W. Peake, 1 S. Price, 1 T.C.M. Morris, 2 A.E. Irvine1 1 2 Queen's University Belfast, BELFAST; Haematology, Belfast City Hospital,
BELFAST, United Kingdom
Background. Previous studies by our group have shown that normal
unstimulated lymphocytes produce a protein which inhibits colony formation
of granulopoietic progenitors, but has no effect on erythroid progenitors.
Therefore, this inhibitor was initially designated GIA (granulopoietic
inhibitory activity). GIA was identified as a glycoprotein of
approximately 30 kDa, with a pI of 7.9-8.4. Furthermore, we demonstrated
that this inhibitor may have physiological significance in that its
production is altered in patients with neutropenia. GIA has proved difficult
to characterise to date since it is produced in relatively low amounts
although it has a high specific biological activity. Aims. Adiponectin is an
adipokine reported to share many of the inhibitory characteristics of
12 th Congress of the European Hematology Association
GIA and has been demonstrated to act as a negative regulator of
haemopoiesis and immune response. This study aimed to determine
whether GIA is adiponectin or if it represents an adiponectin-like molecule.
Methods. Lymphocyte conditioned medium (LCM) from lymhocytes
cultured at 1×10 6 cells/ml in HL-1 minimal medium was used as a
source of GIA. Inhibition of granulopoiesis was tested by co-culturing
LCM with normal bone marrow cells in a myeloid colony assay. Western
blot analysis and ELISA were performed to investigate adiponectin
expression in LCM. RT-PCR analysis of lymphocyte mRNA was carried
out to look for expression of adiponectin at the transcript level. Results.
Inclusion of LCM as 10% of the top layer of agar in a myeloid colony
assay inhibited growth of CFU-GM by 52±11% (n=3), confirming the
presence of the inhibitory activity. Western blot analysis demonstrated
a distinct banding pattern in days 3-7 LCM corresponding to monomers,
dimers, trimers and greater. This is consistent with adiponectin which
circulates as a multimer of trimers. Characterisation of GIA at the transcript
level confirmed that GIA is in fact adiponectin. The N-terminal collagenous
domain, C-terminal globular domain and full length
adiponectin were amplified by RT-PCR analysis and confirmed by
sequencing. Summary and conclusions. Adiponectin is thought to be secreted
exclusively from adipocytes and much of our current knowledge of
this molecule relates to its metabolic functions. Our study provides evidence
that adiponectin is also produced by lymphocytes and may play
a role in the pathogenesis of neutropenia.
0440
ABSENCE OF LEF-1 TRANSCRIPTION FACTOR IN MYELOID PROGENITORS OF PATIENTS
WITH SEVERE CONGENITAL NEUTROPENIA RESULTED IN ABROGATED EXPRESSION OF
NEUTROPHIL ELASTASE AND DEFECTIVE GRANULOPOIESIS
J. Skokowa, 1 J.P. Fobiwe, 2 L. Dan, 2 K. Welte2 1 Medical School Hannover, HANNOVER; 2 Dept. Pediatric Hematology/Oncology
MHH, HANNOVER, Germany
Recently we have shown that Lymphoid-enhancer binding factor 1
(LEF-1) is a decisive transcription factor in granulopoiesis controlling proliferation,
proper lineage commitment and granulocytic differentiation
via regulation of its target genes C/EBPα, cyclin D1, c-myc and survivin.
Expression of LEF-1 and its target genes was abrogated in myeloid progenitors
of severe congenital neutropenia (CN) patients (Skokowa et al.,
Nat Med. 2006;12:1191-7). CN is a heterogeneous syndrome with two
major subtypes: 1) autosomal dominant CN defined by mutations in
ELA2 gene encoding neutrophil elastase (NE) and 2) autosomal recessive
CN (including Kostmann syndrome) carrying HAX-1 mutations, both
characterized by an early stage maturation arrest of granulopoiesis. Interestingly,
in line with LEF-1 levels, ELA2 mRNA expression in myeloid
progenitors as well as NE protein levels in the blood were severely
reduced in CN patients irrespective of ELA2 or HAX1 inheritance. ELA2
gene promoter is positively regulated by direct binding of LEF-1 or
C/EBPα. Transduction of LEF-1-GFP lentiviral constructs containing
cDNA of either full-length or dominant negative (dn) LEF-1 into U937
myeloid cell line led to significant upregulation of ELA2 mRNA and NE
protein, similar as we have shown for C/EBPα. LEF-1 rescue of CD34+
cells of two CN patients resulted in increased NE levels and in granulocytic
differentiation in vitro . Therefore, these data confirm the importance
of LEF-1 in myelopoiesis and in the pathogenesis of CN. Absence
of LEF-1 is a common decisive mechanism of the defective maturation
program of myeloid progenitors in CN downstream of both, ELA2 or
HAX1 mutations.
haematologica/the hematology journal | 2007; 92(s1) | 163