PDF - Advances in Clinical and Experimental Medicine

ORIGINAL PAPERS
Adv Clin Exp Med 2009, 18, 2, 129–133
ISSN 1230−025X
© Copyright by Wroclaw Medical University
LIDIA KARABON1, EDYTA PAWLAK1, ANNA TOMKIEWICZ1, MAREK KIEŁBIŃSKI1,
STANISŁAW POTOCZEK1, DARIUSZ WOSZCZYK4, ANNA JONKISZ3, KAZIMIERZ KULICZKOWSKI1,
IRENA FRYDECKA1,2
Lack of Association Between CD28 Gene Polymorphism
and Multiple Myeloma in a Polish Population*
Polimorfizm genu CD28 a ryzyko zachorowania na szpiczaka mnogiego
w populacji polskiej
1
2
3
4
Department of Experimental Therapy, Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Wrocław, Poland
Department of Hematology, Neoplastic Diseases, and Bone Marrow Transplantation,
Wroclaw Medical University, Poland
Department of Forensic Medicine, Wroclaw Medical University, Poland
Department of Hematology, State Hospital, Opole, Poland
Abstract
Background. Immunophenotypic studies revealed that CD28, a T cell−restricted molecule, is expressed on malig−
nant plasma cells and its expression in multiple myeloma (MM) highly correlates with poor prognosis and disease
progression, while in normal plasma cells CD28 expression is always absent or only weakly positive in small sub−
sets.
Objectives. As dysregulated expression and/or function of CD28 in myeloma cells may be due to polymorphism
of the corresponding gene, this study was undertaken to evaluate the association between CD28c.17 + 3T > C gene
polymorphism and multiple myeloma in a Polish population.
Material and Methods. One hundred fifty patients with MM and 238 healthy subjects were examined. The T >
C transition at position 17 in intron 3 of the CD28 gene was genotyped by polymerase chain reaction followed by
labeling with a SNaPshot kit and detected using a capillary genetic analyzer.
Results. The genotype, allele, and phenotype frequencies did not significantly differ between MM patients and
controls.
Conclusions. The present study was unable to reveal any association between CD28c.17 + 3T > C gene polymor−
phism and the risk of multiple myeloma in a Polish population (Adv Clin Exp Med 2009, 18, 2, 129–133).
Key words: multiple myeloma, costimulatory molecule CD28, gene polymorphism.
Streszczenie
Wprowadzenie. Badania immunofenotypowe wykazały, że CD28, molekuła, której ekspresję stwierdza się głów−
nie na powierzchni limfocytów T, jest również obecna na nowotworowych komórkach plazmatycznych. Jej eks−
presja na nowotworowych plazmocytach w szpiczaku plazmocytowym wykazuje silny związek ze złym rokowa−
niem i progresją choroby. Prawidłowe komórki plazmatyczne zwykle nie ekspresjonują cząsteczki CD28, chociaż
słabą ekspresję tej molekuły czasem obserwuje się na powierzchni niewielkiej frakcji tych komórek.
Cel pracy. Zaburzenia ekspresji lub/i funkcji CD28 na nowotworowych komórkach plazmatycznych mogą być
spowodowane polimorfizmem odpowiedniego genu. Celem pracy było zbadanie zależności między polimorfi−
zmem genu CD28 a ryzykiem zachorowania na szpiczaka plazmocytowego w polskiej populacji.
Materiał i metody. Badania przeprowadzono u 150 chorych na szpiczaka plazmocytowego i 238 zdrowych osób.
Wymiana T > C w pozycji 17 w intronie 3 była genotypowana z użyciem reakcji PCR. Następnie produkty PCR
znakowano techniką SNaPshot i identyfikowano z użyciem sekwenatora kapilarnego.
* This work was supported by a grant no. 1273 from Wroclaw Medical University, Poland.
130
L. KARABON et al.
Wyniki. Częstość występowania poszczególnych genotypów, alleli i fenotypów nie różniła się między grupą cho−
rych na szpiczaka plazmocytowego i grupą kontrolną.
Wnioski. Polimorfizm c.17 + 3T > C genu CD28 nie jest czynnikiem ryzyka zachorowania na szpiczaka plazmo−
cytowego w populacji polskiej (Adv Clin Exp Med 2009, 18, 2, 129–133).
Słowa kluczowe: szpiczak mnogi, molekuła kostymulująca CD28, polimorfizm genu.
Multiple myeloma (MM) is a B−cell malignan−
cy characterized by the proliferation and accumu−
lation of isotype−switched immunoglobulin−pro−
ducing monoclonal plasma cells in the bone mar−
row. Immunophenotypic studies recently revealed
that CD28, a T cell−restricted molecule, is
expressed on malignant plasma cells and that
CD28 expression in MM highly correlates with
poor prognosis and disease progression [1–4].
Increased CD28 expression is seen on myeloma
cells in 26% of newly diagnosed myelomas, 59%
of medullary and 93% of extramedullary relapses,
and 100% of secondary plasma cell leukemias [5]
as well as in all human and murine MM cell lines
[1–3, 6, 7].
Two signals are required for full activation of
T cells. The first, an antigen−specific signal, is sent
via the unique antigen receptor, the T−cell receptor
(TCR/CD3), on T cells. The second signal, termed
costimulation, is independent of the antigen recep−
tor and is critical for allowing optimal T−cell acti−
vation, sustaining cell proliferation, preventing
anergy and/or apoptosis, inducing differentiation
to effector and memory status, and allowing cell−
cell co−operation [8] The main costimulatory mo−
lecule, CD28, is expressed on CD3+ thymocytes,
95% of CD4+ T cells, approximately 50% of CD8+
T cells, and gamma/delta T cells. In normal plasma
cells, CD28 is always negatively or only weakly
positively expressed in a small subset of plasma
cells (< 15%) [4].
In contrast to T cells, little is known about
CD28 function in myeloma cells. Clinical find−
ings suggest that CD28 expression helps MM
cells better survive treatment, which results in
their selective outgrowth [7, 9]. Direct evidence
of a pro−survival effect for CD28 in myeloma
cells has not been reported. However, Qiu et al.
[10] recently found that an anti−CD28 monoclon−
al antibody inhibited cell line proliferation and
induced some morphological signs of apoptosis.
Since dysregulated expression and/or function of
CD28 in myeloma cells may be due to polymor−
phism of the corresponding gene, the present
study was undertaken to investigate the correla−
tion of CD28c.17 + 3T > C gene polymorphism
and the risk of MM.
Material and Methods
The study included 150 unrelated randomly
selected Polish patients with multiple myeloma
and 238 age− and sex−matched healthy subjects.
There were 73 women and 77 men aged 33–87
(median: 66.5 ± 11.06) years. The diagnosis of
MM and stage of disease was established on the
basis of standard clinical and laboratory parame−
ters [11]. The study was approved by the local
ethics committee.
For genotyping the CD28 gene polymor−
phism with a thymine/cytosine substitution at
position of DNA in intron 3 in the CD28 gene on
chromosome 2, the primers F: 5’– CCT GTA
TCA TTT AAT CCA CT–3’ and R: 5’–TGG
AAA AGT TAC ATA AAA CC–3’ were
designed. The designation was based on the com−
plete CD28 gene sequence derived from the
NCBI Sequence Viewer. Allele identification was
achieved by the polymerase chain reaction (PCR)
amplification of 0.5–1.0 ng of genomic DNA
using a Biometra UNO−Thermoblock (Biometra,
Germany). PCR was carried out in a total volume
of 10 µl containing 0.1 µM of each primer and
TaqMasterMix (Qiagen, Germany) with 1 unit
Taq DNA polymerase, 1× PCR buffer (containing
15 mM of MgCl2), and 200 µM of each dNTP.
The PCR profile was as follows: initial denatura−
tion at 95°C for 5 min followed by 30 cycles of
denaturation at 95°C for 1 min, annealing for
1 min at 58°C, and extension for 1 min at 72°C,
and a final extension for 10 min at 72°C. The
amplified DNA was 198 bp in length. The ampli−
fied product for the SNP loci was purified and
minisequenced using a commercial SNapShot kit
(ABI PRISM SNaPshot ddNTP Primer Extension
Reaction Kit, Applied Biosystems, Warrington,
UK) and the primer F: 5’–TCT GGG TAA GAG
AAG CAG CAC–3’. The products of the
SNaPshot reaction were analyzed on an ABI
PRISM 310 Genetic Analyzer (ABI PRISM 310
capillary electrophoresis system) equipped with
GeneScan Analysis Software version 3.1 of PE
Applied Biosystems (USA).
131
CD28 Gene Polymorphisms in Multiple Myeloma
Statistical Analysis
Allele, phenotype, and genotype frequencies
were compared between the groups using
Fisher’s exact test. A p value of < 0.05 was con−
sidered significant.
Results
The patients’ characteristics are shown in
Table 1 and the results in Table 2. The genotype,
allele, and phenotype frequencies did not differ
significantly in the MM patients and controls.
Discussion
CD28 is the only cell surface marker identified
to date that correlates with an advanced state of
multiple myeloma in humans. CD28 is not nor−
mally expressed on B cells [12], but is constitu−
tively expressed on T cells, where it plays an
important role in T−cell activation [8, 13]. The
gene encoding CD28 is located in the chromosome
region 2q33. A CD28 gene polymorphism with
a thymine/cytosine substitution at intron 3 position
Table 1. Patients’ characteristic
+ 17 has been recently identified [14]. The exact
biological role of the CD28c.17 + 3T > C poly−
morphism is not known, but it is hypothesized that
this polymorphism, located near the splice recep−
tor site, may influence the splicing efficiency of
the gene and change the expression of CD28 mol−
ecule. Up to now, CD28 gene polymorphism was
mainly studied in patients with autoimmune dis−
eases. Such studies were performed in thyroid
autoimmune diseases [14], lupus erythematosus
[15], multiple sclerosis [16], autoimmune hepatitis
[17], type 1 diabetes [18], and atopic asthma [19,
20]. The polymorphic frequencies did not differ in
the groups of patients and controls studied. Only
a few studies have been devoted to a possible rela−
tionship between this polymorphism and suscepti−
bility to neoplastic disease [21–24].
Cheng et al. [21] performed genotyping for
CD28c.17 + 3T > C in 62 patients with gastric
MALT lymphoma and 250 unrelated healthy con−
trols. No association between the CD28 gene poly−
morphism and MALT lymphoma was observed. In
the present authors’ previously reported study [22],
no relationship between CD28c.17 + 3T > C gene
polymorphism and susceptibility to cervical can−
cer was found. This result was similar to that
reported by Guzman et al. [23]; however, the fre−
quency of the CD28 (TT) IFN−gamma (AA) com−
bination statistically increased the risk of develop−
Tabela 1. Charakterystyka chorych
Number of patients
(Liczba chorych)
150
Age – median, range
(Wiek – mediana, zakres)
66.5 ± 10.7
Age of diagnosis – median, range
(Wiek – mediana, zakres)
62.0 ± 10.8
Gender – female/male
(Płeć – kobiety/mężczyźni)
73/77
International Staging System
– ISS) [11]
(Stadia kliniczne [11])
Ig subtype
(Typ immunoglobuliny)
Light−chain type
(Rodzaj łańcucha lekkiego)
1
2
3
39%
32%
25%
IgG
IgA
IgM
IgD
Nonse−
cretory
Light−
−chain
disease
others
55%
25%
4%
1%
3%
κ
λ
67%
33%
4.5%
7.5%
Table 2. CD28c.17 + 3T > C genotype, allele, and pheno−
type frequencies in MM patients and controls
Tabela 2. Częstość występowania genotypów, alleli
i fenotypów w miejscu polimorficznym CD28c.17 + 3T >
C genu CD28 u chorych na szpiczaka plazmocytowego
i osób zdrowych
Number/frequency
(Liczba chorych/odsetek)
Genotypes
(Genotypy)
TT
TC
CC
Allele
(Allele)
T
C
Phenotypes
(Fenotypy)
C+
C–
Patients
with MM
(Chorzy na
szpiczaka plaz−
mocytowego)
Healthy
controls
(Osoby
zdrowe)
n (%)
n (%)
P−value
75 (76.5)
21 (21.4)
2 (2.0)
179 (75.2)
55 (23.1)
4 (1.7)
ns.
ns.
ns.
171 (87.2)
25 (12.8)
413 (86.8)
63 (13.2)
ns.
ns.
23 (23.5)
75 (76.5)
59 (24.8)
179 (75.2)
ns.
ns.
132
L. KARABON et al.
ing the disease. In contrast, the CD28c.17 + 3T >
C gene polymorphism influenced susceptibility to
B−CLL, as the present authors found in a study
including 173 B−CLL patients and 336 control
subjects [24]. The presence of the CD28c.17 +
3T > C [C] allele and the CD28c.17 + 3T > C [C]
phenotype increased the odds ratio of B−CLL to
1.59 (p = 0.007) and 1.74 (p = 0.007), respective−
ly. In addition CD28c.17 + 3T > C was associated
with time to Rai stage progression.
To the authors’ best knowledge, no reports
regarding CD28 gene polymorphism and suscepti−
bility to multiple myeloma have been reported so
far. The results of the present study on MM
patients did not demonstrate any differences in
genotype, allele, or phenotype frequencies
between patients and controls. The present data do
not support a role of CD28c.17 + 3T > C gene
polymorphism in predisposition to multiple
myeloma. Therefore, the abnormal CD28 mole−
cule expression on malignant myeloma cells in
patients with MM [1, 3] cannot be related to the
studied CD28 gene polymorphism. It cannot be
excluded that the increased CD28 expression on
malignant cells may be due to the activation of
nuclear transcription factors or abnormalities in
the translation process. It might prove useful to
investigate other immune−system gene polymor−
phisms in MM to identify at−risk individuals in the
population.
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Address for correspondence:
Lidia Karabon
Department of Experimental Therapy
Institute of Immunology and Experimental Therapy
Weigla 12
53−114 Wroclaw
Poland
E−mail: [email protected]
Conflict of interest: None declared
Received: 29.10.2008
Revised: 5.03.2009
Accepted: 23.04.2009