ARTICLE
Auteur(s) :, Giovanni
PORCIELLO1,*, Roberto SCARPATO2, Franca
STORINO3, Lucia MIGLIORE2, Clodoveo
FERRI4, Francesca CAGETTI2, Gabriella
MOROZZI5, Francesca BELLISAI5, Roberto
MARCOLONGO5, Mauro GALEAZZI5
1Servizio di Reumatologia Ospedale Misericordia e
Dolce di Prato, Italy
2Dipartimento di Scienze dell’Uomo e dell’Ambiente,
University of Pisa, Italy
3Istituto di Reumatologia, University of Pisa, Italy
4Istituto di Reumatologia, University of Modena and
Reggio Emilia, Italy
5Istituto di Reumatologia, University of Siena,
Italy
*Dr. Giovanni Porciello. Istituto di
Reumatologia-Policlinico Le ScotteViale Bracci, 53100 Siena, Italy.
Fax (+39) 574/434421.
accepté le 8 Juin 2004
Several studies demonstrate the presence of chromosome alterations,
both spontaneous and induced, in patients with systemic sclerosis
(SSc) [1-10]. But, to our knowledge, only two reports by Emerit
et al. and Galeazzi et al., have studied patients
with Raynaud’s phenomenon (RP), which is often considered one of
the earliest manifestations of SSc [3, 11]. In both studies numeric
and structural chromosome aberrations were observed.Furthermore,
the two above mentioned studies were carried out by conventional
cytogenetic methods such as metaphase analysis of peripheral
lymphocyte cultures while the micronucleus assay is now a widely
accepted tool to easily and accurately detect spontaneous or
induced chromosome damage in circulating peripheral blood cells
[12]. In addition, in order to identify the prevalence of
clastogenic or aneuploidogenic events in micronucleus formation,
fluorescence in situ hybridisation (FISH) analysis is
routinely performed using a pancentromeric DNA probe that
recognises the centromere of all human chromosomes [13].The aims of
the present study were to gain more information on the prevalence
and type of chromosome alterations in RP, to verify whether or not
chromosomal damage is more frequent in the pre-scleroderma RP as
compared to idiopathic RP and, if possible, to establish a
correlation between chromosomal damage and the patient’s
sero-immunological profile. In order to evaluate the prevalence and
type of chromosomal alterations, micronucleus assay and FISH
analysis were used in our study.
Materials and methods
Population
We evaluated chromosomal damage in 30 subjects with
pre-scleroderma RP (all female, mean age
44.0 ± 2.2 years, mean duration of RP
4.3 ± 0.6 years), in 30 idiopathic RP subjects
(all female, mean age 46.0 ± 2.4 years, mean
duration of RP 17.0 ± 5.3 years) and 30 healthy
controls (all female, mean age 47.0 ± 5.2 years).
Careful anamnesis, physical and instrumental examination were
carried out on pre-scleroderma RP subjects to rule out the presence
of any cutaneous alteration (sclerosis, ulcers, telangiectasia,
calcinoses, melanodermia or pseudovitiligo) or visceral alteration
(pulmonary, oesophagus, cardiac or renal) due to SSc.
RP subjects were considered pre-scleroderma when they were
positive for anticentromere antibodies (ACA+) or
anti-topoisomerase-1 antibodies (Sc1 70+) and showed, in nailfold
capillaroscopy carried out with a Videocap 200, one or more
alterations typical of SSc (enlarged loops, giant loops, loss of
capillaries and avascular areas), in the absence of skin or
visceral lesions typical of SSc. Obviously, idiopathic RP and
healthy controls had to be negative for antinucleus antibodies,
have a normal nailfold capillaroscopy, and should not have been in
contact with toxic substances such as benzene, toluene, vinyl
chloride, etc. None of the RP or healthy subjects was taking, or
had taken during the 6 months before the study, potentially
genotoxic drugs; therapy for RP subjects (25 pre-scleroderma
and 26 idiopathic) consisted of vasodilators and rheologic
drugs (buphlomedil, isdradipina and amlodipina). All subjects
studied were evaluated for renal function according to standard
procedures.
The study of antinuclear antibodies (ANA) was carried out by
indirect immunofluorescence (IFI) on HEP-2 cells (Immuno-Concepts,
USA), while anti-ENA were detected by the double immunodiffusion
technique, according to Outcherlony. After the antibody profile had
been characterised, each subject was assigned to one of the
following groups: ANA-, ACA+, or Scl 70+. Only pre-scleroderma RP
subjects underwent further instrumental analysis, such as chest X
Rays, lung function test and alveolo-capillary diffusion of carbon
monoxide (DLCO), ECG, color-doppler-cardiograph ultrasonography,
renal ultrasonography, barium oesophagus X Rays and manometry of
the oesophagus.
Micronucleus test
Micronuclei (MN) are small rounded masses of chromatin organised
like accessory nuclei and visible in the cytoplasm of interphase
cells after one division cycle. Treatment of cultures with
cytochalasin B blocks the cytodieresis of proliferating cells, thus
ensuring an easy recognition of dividing lymphocytes for the
classic binucleated appearance that they assume. From each subject
whole blood cultures were set up in duplicate in medium containing
phytohemagglutinin and incubated at 37°C for 72 h. At
44 h, cytochalasin B was added, and lymphocytes were recovered
according to the classical method [12]. For each subject, MN
frequency was expressed as the number of micronucleated cells
(containing 1 or + MN) per 1 000 binucleate
lymphocytes on a total of 2 000 cells scored.
FISH analysis
Slides containing micronuclei were immersed for 2 min in a
denaturing solution of 70% formamide in 2x SSC (pH 7.0) at a
temperature of around 72°C, dehydrated in a series of cold ethanols
and left to air dry. The digoxigenin-labeled probe (Appligene
Oncor), specific for the centromeric DNA of all human chromosomes,
was denatured at a temperature of 72°C for 5 min and
distributed onto slides left to incubate at 37°C in a moist chamber
overnight. The following day, the slides were washed twice at 37°C
for 4 min in 2x SSC. For immunofluorescence detection,
sequential incubations of 30 min each at 37°C were carried out
with anti-digoxigenin monoclonal antibody (mouse-mouse cellular
hybrids, Boheringer Mannheim), anti-mouse (developed in rabbit),
conjugated with isothiocyanate tetramethylrodamine (TRITC, Sigma),
and anti-rabbit-TRITC (Sigma). The antibodies were reconstituted in
an immunologic buffer of 5% non-fat dry milk in 4x SSC. At the end
of every incubation, the slides were washed for 2 minutes in a
solution of 4x SSC/Tween 20. The slides were then dehydrated via an
ethanol series and left to dry. The unlabeled DNA was
counterstained with 4.6-diamine-2-phenilindol (DAPI) which
developed a blue fluorescence. MN showing or lacking the TRITC
fluorescence signal (red spot) were recorded as C+MN (presence of
whole chromosome) or C-MN (acentric chromosome fragment),
respectively.
A fluorescent signal inside the micronucleus indicates the
presence of whole chromosome(s) (aneuploidogenic mechanism),
micronuclei lacking centromeric signal are thought to be formed by
chromosome fragment(s) (clastogenic mechanism).
We analysed 100 MN per subject and data were expressed, at
group level, as mean percentage of C+MN. Of course, the proportion
of C-MN were represented by the corresponding complementary per
cent values [13].
Statistical analysis
Data management and statistical analyses were performed using SPSS
statistical package. The Chi Square test was adopted for an overall
approach for the comparison of percentages among the groups. When
significance was reached, Fisher’s exact test was used to compare
the frequencies between two groups. Results were also considered
significant using the Chi Square test with Bonferroni’s correction
for the number of comparisons. Spearman’s rank test was used for
the evaluation of the correlations between variables of the various
groups.
Results
Clinical, instrumental and serologic evaluation
In pre-scleroderma RP subjects the mean DLCO was 85% (predict),
while that of pulmonary systolic pressure was 22 mmHg. Renal
function was normal in all subjects included in the study.
Antibody specificity of pre-scleroderma RP subjects was
characterised by 20 patients positive for ACA+ and 10 for
Sc1 70+, while the nailfold capillaroscopy showed the presence of
enlarged loops, diffused micro-haemorrhagies and sub-papillar edema
in 18 cases and giant loops, with a loss of capillaries and
hypertrophy of the papille in other 12 cases. None of the
patients had avascular areas, but a loss of capillaries was present
in most of Sc1 70+ subjects.
None of the idiopathic RP subjects or healthy controls showed
antinuclear antibodies and/or capillaroscopic alterations, and for
this reason they were classified as ANA negative (ANA-).
Cytogenetic analysis
Table I( Table I ) reports the
results of cytogenetic analysis carried out in peripheral
lymphocytes of pre-scleroderma RP, idiopathic RP and healthy
control subjects. The mean frequency of micronucleated cells found
in the pre-scleroderma RP group was statistically higher as
compared to idiopathic RP and healthy control groups, respectively
(37.0 ± 11.5 versus 11.1 ± 3.2 and
10.7 ± 2.7, p < 0.0001). It is very
interesting to note that the idiopathic RP subjects had an MN
frequency nearly close to that of healthy controls.
At the bottom of table I we also report the basal level of
chromosomal damage of RP subjects classified according to the
immunological profile. The ACA+ RP group showed, on average,
spontaneous MN frequency significantly higher as compared to Sc1
70+ RP or ANA- RP groups, respectively (41.0 ± 7.6
versus 25.0 ± 3.5 or 11.1 ± 3.2,
p < 0.0001). No correlation was found between the
duration of RP and the frequency of MN neither between nor within
the RP groups.
The results of FISH analysis are reported in table II(
Table II ). We did not observe any
difference in the percentage of C+MN (or C-MN) between
pre-scleroderma and idiopathic RP subjects
(73.9 ± 7.5 and 72.1 ± 9.2) as compared to
healthy controls (68.8 ± 3.9). Furthermore, C+MN were
found at the same proportion in ACA+ RP, Sc1 70+ RP and ANA- RP
groups (74.3 ± 6.4, 73.6 ± 8.7 and
72.1 ± 9.2).
Table I Chromosome damage (MN test) in peripheral
lymphocytes of Raynaud’s phenomenon (RP) subjects and controls:
results of the micronucleus (MN) test
|
Subject status
|
N° of subjects
|
RP duration (years) mean±S.D.
|
MN frequency (%) mean±S.D.
|
|
Pre-scleroderma RP
|
30
|
4.3±0.6
|
37.0±11.5a
|
|
Idiopathic RP
|
30
|
17.0±5.3
|
11.1±3.2b
|
|
Controls
|
30
|
-
|
10.7±3.5
|
|
eACA+ RP
|
20
|
3.9±0.4
|
41.0±7.6c
|
|
fScl70+ RP
|
10
|
4.7±0.9
|
25.0±3.5d
|
|
gANA- RP
|
30
|
17.0±5.3
|
11.1±3.2
|
aSignificantly different from controls and from
idiopathic RP (p<0.0001).
bNot significantly different from controls
(p≥0.05).
cSignificantly different from Scl70+ RP (p<0.0001)
and from ANA- RP (p<0.0001).
dSignificantly different from RP ANA-
(p<0.0001)
eACA+: anti-centromere antibody positive.
fScl70+: anti-topoisomerase 1 antibody
positive.
gANA-: anti-nuclear antibody negative.
Table II Results of micronucleus (MN) FISH
analysis in peripheral lymphocytes of Raynaud’s phenomenon (RP)
subjects and controls
|
Subject status
|
N° of subjects
|
C+ MN frequency (%) meana±SD
|
|
Pre-scleroderma RP
|
30
|
73.9±7.5
|
|
Idiopathic RP
|
30
|
72.1±9.2
|
|
Controls
|
30
|
68.8±3.9
|
|
bACA+ RP
|
20
|
74.3±6.4
|
|
cScl70+ RP
|
10
|
73.6±8.7
|
|
dANA- RP
|
30
|
68.8±3.9
|
aAll mean values are not significantly different from
each other (p≥0.05).
bACA+: anti-centromere antibody positive.
cScl70+: anti-topoisomerase 1 antibody
positive.
dANA-: anti-nuclear antibody negative.
Discussion
Raynaud’s phenomenon which antedates the appearance of SSc by
several years is generally characterised by the presence of serum
auto-antibodies and nailfold capillaroscopy alterations, whereas in
idiopathic RP the antibodies are absent and nailfold capillaroscopy
is normal [14]. In this context, RP can likely be considered a
pre-scleroderma condition when specific serum antibodies (most of
all anti-centromere and anti-topoisomerase-1 antibodies) and/or
nailfold capillaroscopic alterations (dilations, megacapillaries,
avascular areas) are present. In fact, these features are typical
of SSc and, therefore, are rarely found in suspected secondary RP
to other rheumatic diseases, such as rheumatoid arthritis,
polydermatomyositis, Sjögren’s syndrome, mixed connective tissue
disease, mixed cryoglobulinaemia, etc. [15-20].
In our study, performed by micronucleus assay and by FISH
analysis, we have tested the prevalence and type of chromosomal
damage on cultured peripheral lymphocytes of a group of
30 pre-scleroderma RP subjects, 30 idiopathic RP subjects
and 30 healthy controls. The level of chromosome alterations,
expressed as the frequency of micro nucleated cells, was
significantly higher in pre-scleroderma RP subjects as compared
both to healthy controls and idiopathic RP. When data were analysed
according to the immunological profile, although the case study was
limited, we found the highest spontaneous levels of micronuclei in
ACA+ subjects. Furthermore, as shown by MN fluorescence analysis,
both aneuploidogenic and clastogenic events contributed to MN
formation in the pre-scleroderma RP group (ACA+ and Scl-70+
subjects) at a rate comparable to that of idiopathic RP or control
subjects. In our RP pre-scleroderma subjects, the observed high
levels of chromosomal damage were not induced by pharmacological
therapy, as none of the study subjects assumed potentially
genotoxic drugs.
Our data confirm the results of previous reports in which high
rates of chromosomal anomalies were observed in circulating blood
cells of RP subjects who developed SSc five years later [3] or in
suspected secondary to SSc RP subjects [11] even though in these
studies conventional cytogenetic methods such as metaphase analysis
of peripheral lymphocyte cultures were used. However, it should be
underlined that RP subjects had not been either classified under
immunological and capillaroscopic profile or adequately followed
up. Thus, to our knowledge, the present investigation is the first
one describing a clear link between chromosome damage and the
pre-scleroderma nature of Raynaud’s phenomenon. It is also worth
noting that idiopathic RP subjects have the same very low risk of
displaying chromosome aberrations in their peripheral lymphocytes
as healthy controls. Hence the relevance for looking at chromosome
damage also in RP subjects to distinguish those at risk to develop
SSc. A five year follow up of our patients will allow us to
evaluate the risk of developing SSc in pre-sclerodermic RP.
With regard to the presence of specific auto-antibodies, we
found a clear correlation between ACA and micronuclei formation.
This finding should be confirmed in a larger sample of RP patients
because the literature is still controversial on this topic having
considered only patients with overt SSc [6-10].
Concerning the mechanisms of chromosomal aberrations, it has
been postulated that clastogenic factors may play a role in their
formation. In fact, it is well accepted that serum circulating
clastogenic factors (CF) are present in radiation exposed subjects,
in SSc and in patients with chronic inflammatory disease [21-24].
In SSc, CF would be produced after an increase in oxydative stress,
as recently confirmed by the presence of both increased levels of
circulating antibodies against low density oxydated lipoproteins
and altered respiratory burst, mainly in patients with recent onset
of SSc or at the initial stage of the disease [25-27]. Some classes
of substances were identified as CF components, the tumour necrosis
factor alpha, unusual nucleotides of inosine (ITP), different
cytokines and other oxydant molecules involved in lipid
peroxydation, all with proven clastogenic activity [21-24]. This
activity is probably due to the production of highly reactive
oxygen radicals, especially the superoxyde anion, as ITP is one of
the superoxyde generating components of CF and chromosome damage is
prevented by superoxyde dismutase [28-30].
An alternative mechanism of chromosomal damage might be
represented by the action of specific autoantibodies. As indirect
evidence for the involvement of autoantibodies in inducing
chromosome alterations, IgG antinuclear antibodies were proved to
enter viable cells using mechanisms similar to those of hormones
and growth factors [31, 32]. Thus, the inactivation of the
centromere or the alteration of topoisomerase 1 activity,
enzyme involved in the initial phases of DNA replication and
repair, might cause chromosome malsegregation or the formation of
breakage in the DNA helix, respectively. On the other hand, we
cannot exclude that antibodies could also be the consequence of
unrepaired lesions in DNA considered as non-self.
At present, we are not able to experimentally explain the
mechanisms involved in the high levels of chromosomal alterations
observed in our pre-scleroderma RP group. The results of MN
fluorescence analysis do not support the hypothesis that
antinuclear antibodies are able to directly interact with the
genetic material or chromosome movement to induce the observed
chromosome damage. To demonstrate the role of CF further studies
are needed by treating peripheral blood cultures of healthy
subjects with ultra filtrate plasma from our pre-scleroderma and
idiopathic RP subjects or by evaluating serum level of ITP or other
markers of oxidative stress.
In conclusion, the present study confirms the presence of
elevated levels of chromosome anomalies in peripheral lymphocytes
of pre-scleroderma RP subjects, mainly in those with ACA positive
antibodies. However, FISH analysis would indicate that antinuclear
antibodies are not responsible for the induction of the observed
chromosome damage.
The follow-up of our pre-scleroderma RP subjects, especially
those with the highest MN levels, for 5 years will allow us to
evaluate the risk to develop SSc in this group of patients. Further
studies are also needed to establish whether we could look at the
human micronucleus assay as a diagnostic and predictive tool in RP,
to be added to the other tests, already being used, mainly devoted
to early distinguishing of idiopathic RP subjects from subjects at
risk of developing SSc.
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