| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 From the Department of Ophthalmology, Yokohama City University School of Medicine; the 2 Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Kanagawa; and the 3 Department of Legal Medicine, Shinshu University School of Medicine, Nagano, Japan; the 4 Department of Hygiene and Epidemiology, University of Athens Medical School; the 5 National Tissue Typing Center, George Gennimatas General Hospital, Athens, Greece; and the 6 Newton Wellesley Hospital, Department of Internal Medicine, Boston, Massachusetts.
Abstract
PURPOSE. Behçet’s disease (BD) is known to be associated with HLA-B51 in many different ethnic groups. Recently MICA, a member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), was identified near the HLA-B gene, and a triplet repeat microsatellite polymorphism was found in the transmembrane (TM) region. Because a strong association with BD of one particular MICA-TM allele, A6, was shown in a Japanese population, the present study was conducted to investigate microsatellite polymorphism in Greek patients with BD to know whether this association is generally observed in BD occurring in other populations.
METHODS. Thirty-eight Greek patients with BD and 40 ethnically matched control subjects were examined for MICA microsatellite polymorphism using polymerase chain reaction (PCR) and subsequent automated fragment detection by fluorescent-based technology.
RESULTS. Similar to the Japanese patients with BD, the phenotype frequency of the MICA-TM A6 allele was significantly increased in the Greek patients with BD (50.0% in control subjects versus 86.8% in BD cases), with an odds ratio (OR) of 6.60 (P = 0.0012). The MICA-A6 allele was found in a high frequency both in males and females (weighted OR = 6.68; P = 0.0017). No association was found between the A6 allele and several disease features. A strong association exists between the MICA-TM A6 allele and the B*5101 allele in both the control subjects and patients with BD (weighted OR = 44.39; P = 0.0000023).
CONCLUSIONS. This study revealed in Greek patients a strong association of BD with a particular MICA-TM allele, MICA-A6, providing insight into the molecular mechanism underlying the development of BD.
Behçet’s disease (BD) is a chronic inflammatory disorder with recurrent oral and genital ulcers, uveitis, and vasculitis, along with mucocutaneous, arthritic, and neurologic manifestations.1 It exists worldwide but is found in a higher prevalence in Japan, China, and Korea and along the Silk Route to the countries of the Mediterranean.2 We and others have presented evidence for an HLA association with BD, and HLA-B51 (HLA-B*51 at the DNA typing-defined allele level) was found to be the most strongly associated genetic marker in these populations.2 3 4 5 6 However, it has not yet been clarified whether the HLA-B51 gene itself is the pathogenic gene related to BD or whether it is some other gene in linkage disequilibrium with HLA-B51.
Although the cause and pathogenesis of BD are still uncertain, the
onset of BD is believed to be triggered by the involvement of some
external environmental factors in people with a particular genetic
background. The mean age at onset is the third decade, children are
rarely affected, and few neonatal cases have been reported. The main
microscopic finding at most sites of active BD is immune-mediated
occlusive vasculitis. At the cellular level, CD4+
T cells are found in the perivascular inflammatory exudates, and Th1
cells respond to various stimuli to produce interleukin (IL)-2,
interferon (IFN)-
, and tumor necrosis factor-ß
(TNFß).7
In the recent studies, an increased number of
T cells in the peripheral blood and the involved
tissues, and the phenotypically distinct subset of T cells at
the sites of inflammation were reported.8
9
10
Furthermore,
significant T-cell proliferative responses to mycobacterial
65-kDa heat shock protein peptides and their homologous peptides
derived from the human 60-kDa heat shock protein were observed in
patients with BD.11
12
Therefore, BD is probably not a
simple hereditary disease, and the onset of the disease may be
triggered by some exogenous antigen(s) such as bacteria, virus, or some
microorganism.
Recently, a highly divergent MHC class I chain-related gene
family, MIC, was identified within the class I
region.13
Among the five MIC genes so far
identified, two genes, MICA and MICB, are
functional genes and are located between the HLA-B and
TNF genes. The MICA gene located only 46-kb
centromeric of HLA-B14
is a highly polymorphic
member of this family and is mainly expressed in epithelial cells,
keratinocytes and monocytes15
16
in contrast to
MHC class I genes, which are almost ubiquitously expressed.
The characteristics include the absence of association with
ß2-microglobulin (ß2M), stable expression
without conventional class I peptide ligands, and the absence of a CD8
binding site.16
17
Expression of MICA is not
affected by type I and II interferons,16
17
known to
upregulate markedly the level of typical MHC class I gene
expression. However, notably, the 5'-end flanking region of the gene
for MICA includes putative heat shock elements similar to
those of HSP70 genes, and MICA mRNAs are
augmented in heat shock–stressed epithelial cells.16
17
In the recent study, MICA molecule was found to be
recognized by particular T cells expressing diverse V1
T-cell receptors extracted from intestinal epithelium
tumors.18
It is suggested that MICA molecule
may play an important role as a self-antigen, stress-induced, and may
broadly regulate protective responses by V1 T
cells in the epithelium.18
During nucleotide sequence analysis of the transmembrane (TM) region of
the MICA gene, we found a triplet repeat microsatellite
polymorphism of GCT (alanine).19
Microsatellite
polymorphism was investigated in Japanese patients with BD, and a
strong association of six GCT repetitions (MICA-A6 allele)
with BD was found. Thus, the MICA gene was considered a
strong candidate gene controlling the susceptibility to BD based on its
chromosomal localization, its predicted immunologic function as a
ligand of V1 T cells, its restricted and heat
shock–induced expression in epithelial cells, and a strong association
of this particular MICA-TM allele, MICA-A6, with
BD. In this study, to investigate whether there is the same
MICA association with BD in different ethnic groups,
microsatellite polymorphism in the MICA gene was analyzed in
38 Greek patients with BD and compared with that in 40 ethnically
matched healthy control subjects.
Materials and Methods
Subjects
Patients and healthy control subjects included in this study were
selected from subjects enrolled in a large ongoing case–control study
designed to investigate the risk factors in BD. Thirty-eight Greek
patients with BD (27 men and 11 women), who fulfilled the ISG
(International Study Group) diagnostic criteria for Behçet’s
disease20
based on interview and clinical findings after a
controlled protocol, were included, as well as 40 healthy control
subjects (21 males, 19 females), unrelated to each other or to the
patients and matched to the patients in ethnic origin and age ±5
years. Only white people of Greek ancestry were accepted as either
patients or control subjects. Patients with BD were seen as outpatients
for a period of 1 year, in an outpatient rheumatology clinic in the
Athens metropolitan area and were clinically examined by an experienced
rheumatologist and ophthalmologist. The characteristics of the patients
with BD are presented in Table 1
. The patients’ ages ranged from 20 to 59 years (mean,
37.5 ± 10.6 years). The age of the control subjects ranged from
19 to 68 years (mean, 36.5 ± 12.6 years). All patients and
control subjects agreed to a blood examination conducted according to
the guidelines of the Declaration of Helsinki.
|
Analysis of Triplet Repeat Polymorphism in the Transmembrane Region
of the MICA Gene
For analysis of microsatellite repeat polymorphism in the TM
region of the MICA gene, PCR primers flanking the TM region
were designed.19
The forward primer was labeled at the 5'
end with 6-FAM (PE Biosystems, Foster City, CA), and PCR was performed
according to a protocol described before.19
To determine
the number of triplet repeats in the TM region of the MICA
gene, the amplified products were denatured for 5 minutes at 100°C,
mixed with formamide containing a stop buffer, and electrophoresed on
6% polyacrylamide gels containing 8 M urea in an automated DNA
sequencer (model 373A; PE Biosystems). The number of microsatellite
repeats was estimated automatically using software (Genescan 672; PE
Biosystems) and the local Southern method with a size marker of 350
TAMRA (PE Biosystems) as well as the PCR products of the B-cell lines
used as standard size markers that had been determined for the triplet
repeat polymorphisms by nucleotide-sequence determination, as described
before.19
Statistical Analysis
MICA gene and phenotype frequencies were estimated by
direct counting. Statistical analysis was performed by the
Mantel–Haenszel one-tailed and two-tailed tests.23
The
significance of the distribution of alleles between the patients with
BD and normal control subjects was tested by the
2 method with the continuity correction and
Fisher’s exact probability test. Furthermore, P was
corrected by multiplication by the number of microsatellite alleles or
phenotypes (corrected P: Pc). If a cell frequency
was zero, the odds ratio (OR) was calculated by first adding 0.5 to
each cell frequency.24
To control for the effect of
certain factors, the Mantel–Haenszel weighted OR was
calculated.23
Results
The gene frequencies of the microsatellite polymorphism in the TM region (exon 5) of the MICA gene are shown in Table 2 . All five distinct alleles were found in the control subjects and patients with BD. The MICA-A6 allele was found at a significantly higher frequency among the patients with BD (28.8% in controls versus 64.5% in BD cases; OR = 4.50; P = 0.000016; Pc = 0.000080). All the other alleles were found at a lower frequency (OR <1). Sixteen of the patients with BD (42.1%) and three of the control subjects (7.5%) were homozygotes for the A6 allele (OR = 8.97; P = 0.00046; Pc = 0.0065; Table 3 ). The phenotype frequency of the MICA-A5 and -A6 heterozygote was relatively increased in the patients with BD, but in the other heterozygotes, carrying MICA-A6 on one chromosome was not common. Table 4 shows the phenotype frequencies of the microsatellite polymorphism in the TM region (exon 5) of the MICA gene. Of 38 patients with BD, 33 had the MICA-A6 allele in a homozygous or heterozygous way (86.8%), whereas 20 of 40 healthy control subjects had the A6 allele (50.0%). Thus, the MICA-A6 allele was found to be strongly associated with BD in this Greek sample (OR = 6.60; P = 0.0012; Pc = 0.0059).
|
|
|
|
|
|
|
In this study, we have investigated microsatellite polymorphism in the TM region of the MICA gene in the Greek patients with BD. As a result, similar to the Japanese patients with BD, one particular MICA-TM allele, MICA-A6, was found to be strongly associated with BD in the Greek sample. Thirty-three of 38 patients (86.8%) were homozygous or heterozygous for MICA-A6 (Table 3) , and the MICA-A6 allele was found to be distinctly predominant in the patient group. This MICA-A6 association was widely observed in the Greek patients with BD, regardless of sex and several BD clinical features. Therefore, a specific haplotype(s) surely exists in patients with BD that differs from those in healthy control subjects, and the pathogenic gene related to BD is the MICA gene itself or another gene located very near the MICA gene, including HLA-B or its nearby NOB genes.14
In a previous study we have presented evidence that the BD pathogenic gene is located in the 230-kb segment between the MICB and HLA-C genes.25 26 The MICA gene resides in proximity to the HLA-B gene, only 46 kb from HLA-B, and there was a strong linkage disequilibrium between them. Namely, a strong linkage between the MICA-A6 and HLA-B*5101 alleles was observed in both patients and healthy control subjects (Table 7) . Thus, the HLA-B*510–MICA-A6 haplotype is predominant in the BD patient group and the pathogenic gene responsible for the predisposition to BD should be located on this haplotype. However, the MICA-A6 allele is associated with not only HLA-B51 but also HLA-B44 and -B52, which were not increased in the patient group to any degree. If MICA-A6 is primarily involved in the pathogenesis of BD, the frequencies of the HLA-B44 and -B52 antigens should be higher, along with the increase of this MICA-A6 allele. Further, stratification analysis of the MICA-A6 patients with BD on the possible confounding effect of HLA-B*5101 and vice versa (Table 8) suggests that the significant increase of MICA-A6 in the patient group could be explained by linkage disequilibrium with HLA-B*5101 and that HLA-B*5101 is a primary susceptible locus for BD. The presence of HLA-B*5101–negative patients with BD can be explained by the influence of other genetic factor(s) and/or of various external environmental or infectious agent(s).
However, the possibility of the primary involvement of the MICA gene in the development of BD cannot be fully excluded. The MICA-TM (MICA-A4, -A5, -A5.1, -A6, and -A9) alleles are defined by the number of the microsatellite repeats in the TM region of the MICA gene, and MICA-A6 may not represent a unique MICA allele related to functional significance. In fact, four MICA alleles defined by genetic polymorphism in exons 2, 3, and 4, MICA003, MICA004, MICA006, and MICA009, share the same MICA-TM allele, MICA-A6, as a result of tight linkage.27 One of these four MICA alleles sharing MICA-A6 presumably has a strong association with HLA-B51 and is possibly the real pathogenic gene for BD. In this respect, precise DNA typing in the extracellular domains (exons 2, 3, and 4) of the MICA gene is necessary and is now under investigation in our laboratory.
Although V1 T cells are of unknown
function and no antigens recognized by them have been identified, they
are believed to recognize self-antigens that may be stress-induced. The
MICA molecule that is responsive to heat shock cell-stress
was found to be recognized by V1 T
cells. Thus, two hypotheses can be set forth regarding the primary
involvement of the MICA molecule with BD. First, after some
bacterial infection, local immune response may be induced at the sites
of infection, resulting in production of cytokines followed by
stress-induced expression of MICA. Among many
MICA alleles, MICA-A6 may tend to activate
V1 T cells more effectively through
specific interaction with T cells, because of the presence of
specific amino acids in the a1/a2 domains linked to MICA-A6
or because of a particular V1 T-cell
repertoire that can recognize MICA molecule with
MICA-A6 in an efficient way, thus leading to the onset of
BD. Second, after bacterial infection some bacterial components may
have a specific role similar to that of superantigens in activation of
the MICA molecule. In this model, a bacterial component may
have specifically bound to MICA molecules with the
MICA-A6 allele, induced its expression, and thus may have
increased the MICA-A6 molecules that could activate
V1 T cells, triggering the unusual
immune response responsible for the development of BD.
In conclusion, we have investigated microsatellite polymorphism in the TM region of the MICA gene in a Greek sample and have found a strong association of a particular MICA-TM allele, MICA-A6, with BD. However, it is still uncertain which is the real pathogenic gene responsible for the development of BD, MICA-A6 or HLA-B*5101. In this respect, we have determined the genomic sequence covering the entire 1.8-Mb HLA class I region from the MICB gene to the HLA-F gene (including the MICB, MICA, HLA-B, HLA-C, HLA-E, HLA-A, HLA-G, and HLA-F genes) and have identified more than 700 microsatellite repeats in this region. It is necessary to analyze repeat polymorphisms at these microsatellite loci in patients with BD to determine precise localization of the pathogenic gene related to BD.
Acknowledgements
The authors thank Phaedon G. Kaklamanis for allowing us to use his patients and the patients who took part in this study for their cooperation.
Footnotes
Reprint requests: Shigeaki Ohno, Department of Ophthalmology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
Supported by Grants-in-Aid 07041166 and 08457466 from the Ministry of Education, Science, Sports and Culture, Japan; a grant from the Ministry of Health and Welfare, Japan; and a research grant from Kanagawa Academy of Science and Technology.
Submitted for publication November 24, 1998; revised February 22, 1999; accepted March 10, 1999.
Proprietary interest category: N.
References
+ T cells and natural killer cells in Behçet’s disease J Rheumatol 19,588-592[Medline][Order article via Infotrieve]
receptor from patients with active Behçet’s disease J Rheumatol 21,2301-2306[Medline][Order article via Infotrieve]
T cells in pathogenesis and diagnosis of Behçet’s disease Lancet 347,789-794[Medline][Order article via Infotrieve]
T cells Science 279,1737-1740This article has been cited by other articles:
|
|
 |
|
  R Cohen, S Metzger, M Nahir, and T Chajek-Shaul Association of the MIC-A gene and HLA-B51 with Behcet's disease in Arabs and non-Ashkenazi Jews in Israel Ann Rheum Dis, February 1, 2002; 61(2): 157 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Mizuki, M. Ota, K. Yabuki, Y. Katsuyama, H. Ando, G. D. Palimeris, E. Kaklamani, M. Accorinti, P. Pivetti-Pezzi, S. Ohno, et al. Localization of the Pathogenic Gene of Behcet's Disease by Microsatellite Analysis of Three Different Populations Invest. Ophthalmol. Vis. Sci., November 1, 2000; 41(12): 3702 - 3708. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
