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HLA haplotypes in recurrent aphthous stomatitis: A mode of inheritance?

Article  in  International Journal of Immunogenetics · January 2009

DOI: 10.1111/j.1744-313X.2008.00801.x · Source: PubMed

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HLA haplotypes in recurrent aphthous stomatitis: a mode of inheritance?
Blackwell Publishing Ltd
E. Albanidou-Farmaki,* A. Deligiannidis,† A. K. Markopoulos,* V. Katsares,‡ K. Farmakis† &
E. Parapanissiou†
Summary
The aim of this study was to investigate the genetic
association between recurrent aphthous stomatitis (RAS)
and human leucocyte antigen (HLA) class I and II alleles
and HLA haplotypes. Families selected had at least one
child suffering from recurrent aphthous stomatitis in
addition to one or both of the parents. HLA-A, -B and
-DR alleles were typed in 29 families, 27 nuclear and two
extended (121 subjects). HLA haplotypes of all family
members with RAS were compared with those who were
RAS negative. Although major histocompatibility
complex class I and II gene analysis failed to demonstrate
any significant association between RAS and HLA
antigens, the study of HLA haplotypes revealed a signifi-
cant association between HLA haplotypes and susceptibility
to RAS. The results indicate that susceptibility to RAS
segregates in families in association with HLA haplotypes.
Introduction
Recurrent aphthous stomatitis (RAS) is one of the most
common diseases to affect the oral mucosa and has been
reported as affecting up to 20% of the general population
worldwide at any time (Kleinman et al., 1991). Nevertheless,
there is no study concerning the prevalence of the disease
in the Greek population. The peak age of RAS onset is
during childhood, with a tendency to decrease in severity
and frequency with age (Rogers, 1997).
Although a number of etiologies of RAS have been
proposed, it is generally accepted that the evidence for an
autoimmune response to oral mucosa or to cross-reacting
antigens is strong (Dolby, 1969; Lehner, 1969; Landesberg
* Department of Oral Medicine and Maxillofacial Pathology, School of
Dentistry, Aristotle University, Thessaloniki, Greece,
† Department of Immunology and Histocompatibility, Hippokratio
Hospital, Thessaloniki, Greece and ‡ Department of Genetics,
Development and Molecular Biology, School of Biology, Aristotle
University, Thessaloniki, Greece
Received 3 September 2007; revised 3 September 2007; accepted 31
July 2008
Correspondence: Efthimia Parapanissiou, Director, Department of
Immunology and Histocompatibility, Hippokratio Hospital of
Thessaloniki, Greece. Tel: +30 231 089 2646; Fax: +30 231 081 2957;
E-mail: efipara@yahoo.com
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, International Journal of Immunogenetics 35, 427–432
doi: 10.1111/j.1744-313X.2008.00801.x
et al., 1990). Both humoral and cell-mediated immune
mechanisms to oral mucosal antigens and epithelial cells
have been demonstrated (Graykowski et al., 1966;
Burnett & Wray, 1985; Kayavis et al., 1987a,b; Savage
et al., 1988; Albanidou-Farmaki et al., 1991; Petersen &
Hornsleth, 1993; Regezi et al., 1993; Hasan, 1995;
Kayavis et al., 1995; Vicente et al., 1996; Buno et al.,
1998; Sun et al., 2002; Hasan et al., 2002). The suggestion
of an autoimmune component raised the possibility that
susceptibility to RAS might be genetically determined.
This was supported by the frequency of a family history
in RAS (Lehner, 1968; Davidson & Diamond, 2001).
The human major histocompatibility complex is
known to contribute to the genetic susceptibility to a
variety of diseases. Several reports from different countries
showed a genetic correlation of RAS with HLA antigens,
but the results of these studies are controversial (Mattiuz
et al., 1970; Cavalli-Sforza & Bodmer, 1971; Maxwell,
1971; Lehner, 1972; Mittal et al., 1972; Challacombe
et al., 1977; Lehner et al., 1982).
Taking into account the results of a previous paper of
ours (Albanidou-Farmaki et al., 1988) we carried on the
study in order to analyse HLA haplotypes in families
and to determine if the susceptibility to recurrent
aphthous stomatitis is segregated with certain HLA
allelic haplotypes.
Materials and methods
Subjects
The series consisted of 29 families with at least one child.
At least one of the parents, as well as at least one of the
children suffered from recurrent aphthous stomatitis
(RAS). Twenty-seven of the studied families were nuclear
and two were extended. In the extended families, samples
were available from three generations. All members of the
families were volunteers of Greek origin and nationality
and were recruited from the Department of Oral Medicine
and Pathology at Thessaloniki. All volunteers signed a
special form provided, allowing us to obtain samples from
the members of their families. Samples were obtained
from a total of 121 subjects, representing 60 parents
and 58 children, as well as three grandparents. The mean
age of the children of the nuclear families was 15.4 ±
3.6 years old.
427
428 E. Albanidou-Farmaki et al.
The diagnosis of RAS was made according the criteria
of Lehner (1972), and all were diagnosed as having minor
or major RAS. Subjects with herpetiform ulceration or
those in whom the diagnosis was not clear were excluded
from the study. Although most subjects had minor RAS,
it was noted that some children suffered from major RAS,
while the diagnosis in the parent was minor RAS and vice
versa. However, no distinction was made in this study for
the two types of RAS. From the 121 subjects, the 76
suffered from recurrent aphthous stomatitis, while the
remaining 45 were healthy.
HLA typing
Patients and all members of the families were typed for
HLA class I and class II alleles. HLA-A, -B and -DR typing
was performed using the polymerase chain reaction (PCR)
with sequence-specific primers (PCR-SSP) low resolution
method.
Genomic DNA was extracted from 10 mL peripheral
blood with 0.1% EDTA anticoagulant by the salting-out
method (Kimura & Sasasuki, 1991). PCR-SSP materials
were provided commercially (Collaborative Transplant
Study).
The nomenclature of PCR-SSP typing is based on the
HLA Nomenclature Committee of the Twelfth Inter-
national Histocompatibility Workshop and Conference.
Statistical analysis
Haplotype frequencies of the patients with RAS were
calculated. All pedigree data were organized in 29 families
(27 nuclear and 2 extended). Multi-transmission data
disequilibrium test (multi-TDT) based on logistic regression
for use with multiallelic markers was carried out using the
statistical software multtdt.exe (Abecasis et al., 2000).
One thousand permutations were performed to evaluate
the statistical significance. The three HLA genetic loci (i.e.
HLA-A, B, DR) were found to have 15, 24 and 10 alleles,
respectively, in the studied sample. Multi-TDT was
selected, due to the fact that it can be performed with
more than two multiallelic genetic loci, as those in the
present study, contrary to the sib-pair analysis in which
the presence of at least two affected children is necessary.
Results
In all, 29 different composite HLA haplotypes (for HLA-
A, -B, -DR) were found in the RAS patients, of which the
HLA haplotype A*03B*07DRB1*13 was the most
common with a frequency of 6.58%. Susceptibility to
RAS was not obviously associated with any particular
haplotype, but it clearly segregated in some families with
HLA haplotypes (Figs 1 and 2).
Multi-TDT analysis was conducted on families with
affected children. The results are presented in Table 1. The
first locus refers to HLA-A, the second to HLA-B, and the
third to HLA-DR. The significance level is set to 95%.
The multiparameter test (including all three loci) indicates
Journal compilation © 2008 Blackwell Publishing Ltd, International Journal of Immunogenetics 35, 427– 432
Table 1. Statistical transmission data disequilibrium (TDT) and P-values
among human leucocyte antigen (HLA)-A, -B and -DR loci. Locus 1
stands for HLA-A, locus 2 for HLA-B and locus 3 for HLA-DR. The
asterisk (*) indicates the statistically significant values
All studied HLA loci Two-loci system
TDT value P Stat. value P
Locus 1 and 2 77.060 0.361
Locus 2 and 3 91.928 0.019*
Loci 1–3 147.692 0.002*
deviation from linkage disequilibrium (P = 0.002), which
is indicative of the residence of RAS responsible genetic
locus within all three studied HLA genetic loci. Further-
more, the TDT analysis on two loci system (i.e. between
HLA-A & HLA-B and HLA-B & HLA-DR) indicates that
the responsible genetic locus for RAS is probably located
between the second and the third genetic loci (i.e. between
HLA-B and HLA-DR) (P2–3 = 0.019), while between the
first and the second genetic loci (i.e. HLA-A and HLA-
B) there is no statistically significant difference (P1–2 =
0.361).
Discussion
This is the first study of HLA allelic haplotypes in a Greek
population group suffering from RAS. Family studies
were conducted in order to examine the possible mode of
inheritance of the susceptibility genetic loci for RAS, in
accordance to HLA allelic haplotypes.
The use of family data in the transmission/disequilib-
rium test (TDT) permits the study of the transmission of
haplotypes that extend over several adjacent markers.
Descriptive analyses have shown that such data may
produce more-convincing evidence of association, by
identifying ancestral marker haplotypes (Clayton & Jones,
1999).
In the present study there was an apparent relationship
between the inheritance of HLA haplotypes within
families and RAS. This strongly suggests that susceptibility
to RAS segregates with a gene residing in the small arm of
chromosome 6, where the studied HLA genetic loci are
located. Within the studied families, there was a striking
concordance between the possession — as well as the
inheritance — of a specific HLA haplotype and the
susceptibility to RAS. In fact, this was apparent in 43 of 58
children, after the index case had been verified. However,
it is still possible that the remaining child might also
develop RAS. All children of the studied families were
15 years or older, as the majority of RAS cases (over 95%)
are developed by the age of 20 years.
The multi-TDT analysis suggests that the responsible
genetic locus for the RAS is located among the three HLA
loci (P = 0.002). Furthermore, the same analysis examining
only two HLA loci each time, indicates that the responsible
locus for RAS is probably located between the second and
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, International Journal of Immunogenetics 35, 427–432
© 2008 The Authors

HLA haplotypes in RAS

Figure 1. (a & b) Human leucocyte antigen (HLA) allelic haplotypes (HLA-A*, -B*, -DRB1*) in the 27 nuclear families. Bold face indicates the susceptibility haplotype for recurrent aphthous stomatitis (RAS).

429
430 E. Albanidou-Farmaki et al.
Figure 1. Continued
the third HLA genetic loci (i.e. between HLA-B and HLA- Co-segregation between HLA antigen haplotypes and
DR) (P2–3 = 0.019, while P1–2 = 0.361). RAS appeared to be true either for the major, minor or
Previous studies that had examined the relationship herpetiform types of the disease. A similar association
between HLA and recurrent aphthous stomatitis had between A2B12 antigen haplotype and the responsible
variable results. genetic locus has also been reported for the mucocutaneous
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, International Journal of Immunogenetics 35, 427– 432

Figure 2. Human leucocyte antigen (HLA) allelic haplotypes (HLA-A*, -B*, -DRB1*) in the two extended families. Bold face indicates the susceptibility
haplotype for recurrent aphthous stomatitis (RAS).
form of Behçet’s syndrome (Lehner et al., 1982). Close
association between HLA-DR5 antigen and RAS, as well
as strong positive linkage disequilibrium with HLA class
I antigens for some haplotypes has been revealed in a
study examining the distribution of HLA-A, -B, -C
and -DR antigens in 106 Greek parents with and 120
controls (Albanidou-Farmaki et al., 1988).
On the other hand, Dolby (1969) and Platz et al. (1976)
were unable to establish an association between HLA-A
or -B antigens with recurrent aphthous stomatitis.
Another study reported the presence of antigen HLA B51
in six of 26 subjects with RAS and the presence of antigen
CW7 in other six of 26 subjects.
Since HLA B51 antigen is raised in some series of
patients with Behçet’s disease (Verity et al., 1999), recogni-
tion of HLA antigen haplotypes in RAS may be predictive
of subsequent development of Behçet’s disease (Sun et al.,
2001), although others have produced evidence against a
common disease spectrum between RAS and Behçet’s
syndrome (Porter et al., 1998).
Previous cross-sectional studies from the UK and
Greece have suggested that certain HLA antigens are linked
to RAS, notably the class I antigens A2 and B12 (Lehner
et al., 1982) and the class II antigen DR5 (Albanidou-
Farmaki et al., 1988), although associations have not
been found in all populations examined (Platz et al.,
1976; Verity et al., 1999). In the current longitudinal
study, associations between individual HLA alleles and
RAS were not found. However, it has been estimated
that several hundred genes reside in the HLA region, and
thus it is possible that the gene responsible for susceptibil-
ity to RAS is not directly an HLA locus, but a locus that
is in linkage disequilibrium with the HLA loci.
The susceptibility genetic loci for RAS, possibly Ir
genes, have a prevalence of about 20% in the general
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, International Journal of Immunogenetics 35, 427–432
HLA haplotypes in RAS 431
population; however, in specific population groups it
might reach up to 60% (Burnett & Wray, 1985; Kleinman
et al., 1991; Albanidou-Farmaki, 2000). In a previous
study of ours, there was a significant increase (P < 0.05) of
HLA A1DR5, A1CW4 and A2CW2, A2B12DR5 antigen
haplotypes frequency in patients with RAS in a Greek
population sample than in a control group without RAS
(Albanidou-Farmaki et al., 1988, 1990). This finding
supports the hypothesis of a specific antigen haplotype is
possibly responsible for the disease. In the current study,
it was of interest to determine whether there was evidence
of inheritance of any such resistance gene analysed in the
same way as for the susceptibility gene.
In conclusion, the present family study strongly suggests
that susceptibility to RAS segregates with specific HLA
haplotypes. However, since a number of different haplo-
types were involved, multi-TDT analysis indicates that the
susceptibility to RAS is segregating with a genetic locus in
chromosome 6, which is probably resides between HLA-
B and HLA-DR genetic loci and not between HLA-A and
HLA-B loci.
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© 2008 The Authors