ARTICLE
Alopecia areata (AA) is an organ-specific autoimmune disease directed
against the hair follicle [1, 2]. As is the case with many other autoimmune
diseases, there is an association between AA and HLA. The most significant
associations between AA and HLA loci vary with different reports from
different populations as HLA antigens are population specific [2]. There
is a strong association of AA with certain HLA class II alleles. Class
II DRB1 alleles, HLA DR4 and HLA DR5, were found to be increased in Danish
[3], Italian [4], English [5] and American patients [6, 7]. HLA DR7 was
increased in Russian AA patients [8]. It was reported that DQB1*03 and
DRB1*1104 alleles are markers of general susceptibility to AA [7, 9-11].
In addition, the frequency of two other alleles, DRB1*0401 and DQB1*0301,
is significantly increased among patients with alopecia totalis (AT) and
those with alopecia universalis (AU), most extensive forms of AA.
We aimed to investigate HLA class II DR and DQ DNA typing in Turkish
patients with AA, and to investigate the relation of HLA Class II DNA
profile with the age at onset and severity of the disease.
Material and methods
Patients and controls
Sixty-five unrelated patients with AA (42 males and 13 females) aged
19 to 57 years were included in this study. Fifty healthy transplant donors
(36 males 14 females) were used as controls. Demographic data were gathered
from all patients and including age at onset and presence of other autoimmune
diseases. The patients were characterized according to alopecia areata
investigational assessment guidelines [12]. The proportion of scalp hair
loss was determined by mentally dividing the scalp into 4 quadrants and
estimating the percentage of scalp surface that all the alopecic areas
would occupy if placed together [12]. The total group of AA patients as
well as various subgroups according to scalp hair loss were compared to
the control group. First group (n = 65) included all alopecia areata patients,
second group (n = 31) was the patients with less than 25% scalp hair loss,
third group (n = 12) was the patients with scalp hair loss of 25 to 75%
and fourth group (n = 22) was the patients with AT, AU or AT/AU. There
were no patients with scalp hair loss of 75 to 99%. These groups were
compared with the control group. In addition, patients were divided into
two groups based on age at onset of the first episode of disease: age
up to 12 years (early onset) and older than 12 years (late onset).
DNA extraction and PCR amplification
DNA extraction was carried out by the classical phenol-chloroform method
from fresh whole blood samples which anticoagulated by EDTA. Polymerase
chain reaction/ sequence specific primer (PCR/SSP) method was used to
determine HLA DNA typing. A dried primer stock solution consisting of
an HLA specific primer mix, i.e., allele and group specific primers
and internal positive control primer pairs, was aliquoted in 0.2 ml PCR
tubes. To check PCR conditions, the internal positive control primer pairs
that amplify the human growth hormone gene segments were used in each
specimen. The PCR master mix contains: 0.4 U/mul Taq Polymerase, 200 muM
dNTPs, 50 mM KCL, 1.5 mM MgCl2, 10 mM Tris-HCl pH 8.3, 0.001%
w/v gelatin, 5% glycerol, 100 mug/ml cresol red at final concentration.
Amplification reaction contains: 2 mul template DNA (20-100 ng/ml), 3
mul PCR master mix, 5 mul nuclease free dH2O for one SSP reaction.
PCR cycling parameters: denaturation at 94° C for 2 min; 1 cycle,
denaturation at 94° C for 10 sec, annealing and extension at 65°
C for 60 sec; 10 cycle, denaturation at 94° C for 10 sec, annealing
at 610° C for 50 sec, extension at 72° C for 30 sec; 20 cycle,
as the manufacturer described (HLA DR&DQ Combi SSP, Olerup SSP
AB, Sweden). PCR products run 2% (w/v) agarose gel electrophoresis.
Presence and relative lengths of the specific PCR products were interpreted
with Helmberg-Score interpretation Software (GenoVision Inc., West
Chester, PA. USA).
Statistical analysis
The frequency of DR and DQ alleles from patients and controls were compared
using chi square analysis of 2 x 2 tables and Yates' correction, and strength
of associations was estimated by odd ratio (OR) of chi square determination
with use of the EPI-INFO 6 statistical program. If five or fewer persons
were present per group, Fisher's exact two-tailed test was used [7]. P
values for DRB1*03 allele were corrected by multiplying by the number
of alleles tested (19 alleles). P values for DQB1*03 and DRB1*04 alleles
were not corrected because they have been previously reported to be significantly
associated with AA.
Results
The frequencies of HLA DR and DQ were shown in Table
1. The frequency of HLA DQB1*03 allele in all patients was significantly
increased to 86.1% as compared to 62.0% in controls (P = 0.005, OR = 3.81).
The frequency of DRB1*03 in all patients was significantly (uncorrected
P = 0.01, OR = 0.17) decreased to 4.6% as compared to 22.0% in controls,
but these negative associations of DRB1*03 were not statistically significant
when corrected.
When the second group (scalp hair loss less than 25%) was compared to
control group; the frequency of DRB1*03 was significantly decreased in
this patient group (3.2% versus 22.0%, uncorrected P = 0.02, OR
= 0.12) but not statistically significant for this allele after correction.
When the group of patients with 25-75% scalp hair loss was compared
to control group; the frequencies of DRB1*04 alleles was significantly
increased to 66.7% as compared to 28.0% (P = 0.02, OR = 5.14).
When the AT, AU or AT/AU group was compared to control group; DQB1*03
allele was associated with an increased frequency (90.9%) in AT, AU or
AT/AU patients versus controls (P = 0.03, OR = 6.13). There were
no significant differences for the other DQ alleles and the DR alleles
tested between patients and controls.
When patients with early onset were compared to patients with late onset;
no significant difference in allele/or alleles were found. In the group
of hair loss less than 25%, the percentage of patients with early onset
was 10%. In the group of hair loss of 25% to 75% and in the group of AT,
AU or AT/AU, the percentages of patients with early onset were 42% and
55%, respectively. Among the 65 patients, 8 had a family history of AA,
3 had a family history of psoriasis vulgaris, 1 had a family history of
myasthenia gravis, 1 had a family history of vitiligo and 2 had a family
history of diabetes. Two patients had vitiligo, and 3 patients had allergic
rhinitis. Thyroid antimicrosomal antibody determinations were performed
in 15 patients with AT, AU or AT/AU. The levels of these antibodies were
increased in 8 patients. The disease had been present for a range of two
months to 37 years in all patients.
Discussion
Our results suggest that DQB1*03 antigen is a marker for general susceptibility
to AA in the Turkish population. This antigen was not significant in any
form of patchy AA, but significant in the severe form of AA (AT, AU or
AT/AU). Therefore, DQB1*03 allele may also serve as special genetic marker
for susceptibility to the severe form of AA. This is the first study by
biomolecular methods investigating HLA class II alleles in Turkish patients
with AA. Kavak et al. [13] recently reported a significantly higher
frequency of HLA-A1, B62, DQ1 and DQ3 antigens in Turkish patients with
AA. However their study was performed by serological method. They found
that the frequency of DQ3 antigen was 15.9% in patients with AA, but we
found a higher frequency of DQB1*03 allele (86.1%) in patients with AA.
DQ alleles associated with severe persistent AU/AT may be contributing
to susceptibility and/or to failure of suppression of the immune reaction
when initiated [2]. This could be related to the fact that immunologic
suppression by CD8+ T cells is dependent upon use of the T-cell
receptor and is HLA-DQ restricted [14]. Many studies have been performed
on the association between AA and HLA [3-10, 13, 15-17]. Colombe et
al. [9] typed for HLA-DR and -DQ antigens in 283 American patients.
They found a statistically significant increase in frequency of four specific
HLA alleles, namely DQB1*03 (DQ3); DQB1*0301 (DQ7), a subtype of DQB1*03;
DRB1*1104 (DR11); DRB1*0401 (DR4). In this study, DQB1*03 antigen was
significant for both the patchy AA (79.2%) and AT/AU (87.6%) groups, as
compared to control (48.2%), whereas DQB1*0301 was significantly elevated
only in the AT/AU patients. In addition to DQB1*0301, DRB1*0401 was found
with a significantly increased frequency uniquely in the AT/AU group of
patients, suggesting that these two alleles might serve as genetic markers
for susceptibility for the more severe manifestations of AA in USA. They
also found an increase in the frequency of DRB1*1104 (DR11) in all groups
of AA (patchy AA, AT/AU and mixed groups). They suggested that DQB1*03
and DRB1*1104 served as markers of general susceptibility to AA. Moreover,
Welsh and colleagues reported the same 80% incidence of DQB1*03 in a group
of 85 American AA patients [7]. Supporting these two studies, we have
also found a general association of DQB1*03 with AA.
Twenty-two Danish patients with AA showed an increased frequency of
DR4 (uncorrected P = 0.04) [3]. Orecchia et al. [4] found an increased
frequency of DR4 in Italian AA patients, and Duvic et al. [6] reported
a positive correlation with DR4 in a large American white population of
patients with AA, but in these three studies p values were not significant
when corrected for the number of HLA antigen tested. A significant increase
of DR4 (50% compared to 26% in controls; corrected P = 0.04) in 54 British
Caucasian AA patients was also reported by Zhang et al. [5]. The
frequency of DR4 was only increased in the group of scalp hair loss greater
than 25% and less than 75% in our study. Furthermore, Duvic et al.
[6] found that the frequency of DR3 was decreased in patients with AA
relative to controls, but this decrease was not significant. As in this
study, the frequency of DRB1*03 was decreased in all AA patients and the
group of hair loss less than 25%, but this negative association was not
significant when corrected. On the other hand, DQB1*06 was decreased relative
to controls (56%) in all patients (32%, OR = 0.37, corrected P = 0.0045)
in the study by Welsh et al. [7], but in our study there was not
such a significant negative association for this allele.
When we look at the age at onset of disease and HLA alleles distribution,
in AA there was no significant difference in the frequency of HLA alleles
between early onset and late onset patient groups. Orecchia et al.
[4] found a strong correlation between age of onset and severity of disease
with the presence DR5 (DR11) in a study of Italian AA patients. However,
in our study the more severe form of AA was associated with early onset.
There is a high frequency of a family history of AA in affected persons,
ranging from 10% to 42% of cases [1]. The family history of AA was 12.3%
in our study, in keeping with previous reports.
Article accepted on 29/1/02
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International Short Course
on Dermoscopy
July 16-20, 2002
Department of Dermatology
University of Graz
Graz, Austria
This course is designed for all colleagues interested
in learning dermoscopy for diagnosing and managing equivocal pigmented
skin lesions more effectively. Special emphasis will be given to
correlating meticulously the clinical and dermoscopic images of
pigmented skin lesions with its underlying histopathologic findings.
Academic Directors
H. Peter Soyer, M.D., Professor of Dermatology,
Department of Dermatology, University of Graz, Graz, Austria.
Giuseppe Argenziano, M.D., Assistant Professor
of Dermatology, Department of Dermatology, Second University of
Naples, Naples, Italy.
The detailed programme will be presented
on the course website : http://dermoscopy.uni-graz.at
For further information please contact:
cme.dermoscopy@uni-graz.at
|
CONCLUSION In
conclusion, DQB1*03 allele is a marker for general susceptibility to AA
and may also serve as special genetic marker for susceptibility for the
more severe form of AA in our population as well. However, this association
is not related to the age at onset of the disease. REFERENCES
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areata. J Am Acad Dermatol 1986 14: 129-30.
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