|Molecular Vision 2003;
Received 29 April 2003 | Accepted 28 August 2003 | Published 2 September 2003
MICA association with presumed ocular histoplasmosis syndrome (POHS)
Judith Reinders,1 Erik H.
Rozemuller,1 Jenny V. Ongkosuwito,2 Martine J. Jager,3
Marcel G. J. Tilanus,1 Maria S. A.
1Department of Pathology, University Medical Centre Utrecht, Utrecht, The Netherlands; 2Department of Ophthalmology, Academic Medical Centre Amsterdam, Amsterdam, The Netherlands; 3Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands; 4Department of Ophthalmology, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Correspondence to: Dr. M. G. J. Tilanus, Department of Pathology, H04.312, University Medical Centre Utrecht, Heidelberglaan 100, P. O. Box 85500, 3508 GA Utrecht, The Netherlands; Phone: +31-(0)30-2507665; FAX: +31-(0)30-2544990; email: M.Tilanus@azu.nl
Purpose: MHC class I chain related gene A (MICA), a polymorphic and stress-inducible cell surface molecule, is located centromeric to human leukocyte antigen locus B (HLA-B) in the human leukocyte antigen (HLA) region on chromosome 6. MICA is thought to be involved in the innate immune response. An alanine repeat polymorphism is present in the MICA transmembrane region, for which several disease associations have been reported. Previous research indicated an association with the HLA-B7-DR2 haplotype. In this study we investigated the association of the polymorphic MICA alanine repeat and the ocular disease presumed ocular histoplasmosis syndrome (POHS).
Methods: Twenty-four patients and 106 controls were evaluated for the alanine repeat. A PCR reaction was performed to amplify the polymorphic MICA alanine repeat. Allele lengths of the MICA alanine repeat in patients and controls were determined with GeneScan analysis.
Results: No significant associations were observed. Phenotype frequencies of the polymorphic MICA alanine repeat were not significantly different between POHS patients and controls. Neither in the complete patient group compared with the control group nor in one of the subdivided patient groups compared with the control group.
Conclusions: We conclude that the MICA alanine repeat is not a disease-associated factor in POHS. Further analysis of other genes in the B-DR region might elucidate the association of POHS with B7-DR2.
Several syndromes in uveitis are known as white dot syndromes. One of them is presumed ocular histoplasmosis syndrome (POHS), a well characterized entity consisting of white atrophic circumscribed scars scattered throughout the fundus, disciform macular scars, peripapillary scarring, and the absence of signs of vitreous inflammation. Presumed ocular histoplasmosis syndrome (POHS) occurs in young adults and is seen more often in females . POHS can be discriminated from other ocular diseases, e.g. punctate inner choroidopathy (PIC), using well defined criteria [2,3].
In the past, the fungus Histoplasma capsulatum was thought to have an aetiological role in this syndrome, this assumption was based upon epidemiological data . Other studies have shown an association with the HLA-B7-DR2 haplotype on chromosome 6 [5,6]. Recently, this correlation has been refined with human leukocyte antigen (HLA) high resolution typing  and extended criteria to define POHS in a group of Dutch patients in which Histoplasma capsulatum is not endemic . Among the HLA-B7-DR2 haplotype, also associated in the Dutch patients, the association directed more significantly to HLA-DR15, a subgroup of HLA-DR2. HLA high resolution typing enabled us to identify all the individual amino acids of the HLA alleles. In this patient group, a strong association was observed with the amino acid isoleucine at position 67 of the HLA-DRB molecule . However this could not explain fully the association. The HLA region is highly polymorphic  and is not only composed of HLA genes, but also of many HLA-related and non-HLA related genes [8,9]. Many immune response genes are located in the HLA-B-DR region. One of the candidate genes in this region is the HLA-related gene MHC class I chain related gene A (MICA). Research groups have reported several disease associations with the MICA alanine repeat polymorphism: ankylosing spondylitis , Behçet's disease [11-13], ulcerative colitis , psoriasis , and psoriatic arthritis .
The MICA gene is located centromeric to HLA-B on the short arm of chromosome 6 and some MICA alleles are in linkage disequilibrium with HLA-B [17,18]. MICA is considered to be a stress-inducible surface molecule. The molecule does not interact with beta-2-microglobuline and probably does not bind any peptide. Immune responses independent of peptide presentation fit in the concept of immune responses in POHS since Histoplasma capsulatum is not endemic in Dutch patients. MICA molecules can be recognised by alpha-beta CD8+T-cells, gamma-delta CD8+T-cells, and natural killer (NK) cells bearing the NKG2D receptor [19,20]. Therefore the MICA molecule is thought to be involved in the immune response, either by activating NK cells or by costimulation of T cells [19,21,22]. An alanine repeat polymorphism is found within exon 5 of the MICA gene, encoding the transmembrane region (TM), resulting in different numbers of alanine repeats in this region. Seven different alleles can subsequently be found: MICA-A4, -A5, -A5.1, -A6, -A7, -A9, and -A10 allele [23,24]. The MICA-A5.1 allele has one extra nucleotide in the transmembrane region compared with the MICA-A5 allele, leading to a frame-shift. This results in a premature stopcodon within the transmembrane region and as a consequence no cytoplasmic tail is present in these MICA molecules which might lead to an aberrant expression on the cell surface. Although the function remains to be established it has recently been shown that MICA-A5.1 molecules are aberrantly transported, to the apical membrane of intestinal epithelial cells instead of to the basolateral membrane, the site where T cells and NK cells are present. The functional relevance of this A5.1 is not yet elucidated but no MICA expression on the cells might result in an escape from immune surveillance by T and NK cells . MICA protein expression is observed not only in normal cells like gastrointestinal epithelium cells , endothelial cells, fibroblasts, keratinocytes and monocytes  but also in epithelial tumor cells . Due to the polymorphic character of the HLA region, its genes and their presumed immunological function, MICA is a good candidate gene in the B-DR region to be used in disease-association studies.
The aim of our study was to investigate whether the MICA transmembrane polymorphism is associated with POHS. To clarify the involvement of MICA in POHS, the distribution of the MICA alanine repeat polymorphism was analysed in Dutch POHS patients and Dutch blood transfusion donors (controls). All patients and controls gave their informed consent for their participation in this study.
Patients and controls
Twenty-four patients diagnosed with POHS were selected in the period 1995-1999 from the University Ophthalmic Department of Utrecht and Amsterdam, the Netherlands. Patients were only included if both fundus photography and data concerning the ophthalmic examination and therapy were available. Fundus photography of 24 patients (18 female and 6 men) were judged in a masked fashion after being mixed with 24 photographs of patients with similar ocular findings. Criteria for the diagnosis of POHS were the presence of peripapillary atrophy, punched out peripheral lesions (histospots) and the presence of a macular scar or subretinal neovascularisation. Two groups of patients were defined based on the three forementioned criteria. Group 1 consisted of patients who met all three criteria, the complete POHS group (POHS score=3), and group 2 consisted of patients who only met 1 or 2 criteria, the incomplete POHS group (POHS score=1 or 2). If any sign of ocular inflammation, e.g. vitreous cells, was present at any time in patients, patients were excluded from this study since they did not meet the criteria for POHS as mentioned before. A venous blood sample was given by the patients if included in the study. As controls 106 healthy Dutch blood donors were randomly selected. From both patients and controls genomic DNA was isolated from peripheral blood with the salting-out method previously described . The age of each patient was determined at the timepoint when they were diagnosed with POHS. The mean age for the complete POHS patient group (POHS score=1, 2, or 3) was 32 years. The subdivided POHS group 1 (POHS score=3) and POHS group 2 (POHS score=1 or 2) had a mean age of respectively 32 years and 31 years. The mean age of the control persons was 34 years and is not significant different from the patients.
PCR amplification of MICA alanine repeat
The alanine repeat was amplified by polymerase chain reaction (PCR) using primers flanking the repeat, the forward primer, MICA5F (5'-CCTTTTTTTCAGGGAAAGTGC-3'), labeled at the 5' end with 6-carboxyfluorescein amidite (6-FAM) and the reversed primer, MICA5R (CCTTACCATCTCCAGAAACTGC; Eurogentec, Maastricht, The Netherlands). The MICA5F primer is located on the intron 4/exon 5 boundary, while the MICA5R primer is located in intron 5 . The PCR amplification was carried out in a total volume of 50 μl, containing 100 ng genomic DNA, 10μl of 10X PE II PCR buffer (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), 0.2 μl of AmpliTaq Polymerase (5 units/μl; Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), 6 pmol of each PCR primer, 3 μl 25 mM MgCl2, (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) and 1 μl 10 mM dNTPs (Invitrogen, Breda, The Netherlands). In a thermal cycler (Perkin Elmer Cetus 480) DNA was amplified starting with a denaturation step for 4 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 56 °C and 45 s at 72 °C and ending with 5 min at 72 °C and hold at 4 °C. A layer of mineral oil was placed on top of the PCR mix to avoid evaporation during the PCR program.
GeneScan analysis of the MICA alanine repeat
To determine the number of repeats present in each sample, fluorescent labeled PCR products were mixed with TAMRA 500 GeneScan size standard (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) and loading buffer, containing deionized formamide, 1X TBE and Dextran Blue. Products were denatured at 95 °C for 2 min and subsequently electrophoresed on a 4.25% polyacrylamide GeneScan gel in an ABI 377 DNA sequencer (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). The gel is composed of 18 g urea (Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands), 5.4 ml 40% acryl/bisacrylamide (19:1; Biorad, Veenendaal, The Netherlands), 5.0 ml 10X TBE and 25 ml MQ. The urea was solved by heating and mixing the solution, subsequently filtered through a 0.45 μm filter and MQ was added up to a total volume of 50 ml. After adding of 250 μl 10% APS (Biorad, Veenendaal, The Netherlands) and 25 μl TEMED (Biorad, Veenendaal, The Netherlands) the gel polymerized. Determination of the number of repeats was performed by analyzing the GeneScan data using GeneScan Analysis software 3.1.2 and Genotyper software 2.5.2 (both from Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands).
Statistical analysis of MICA alanine repeat
Phenotype and genotype frequencies were estimated by direct counting. The statistical significance of the phenotype and genotype frequency of the different MICA alleles between patients and controls was evaluated by Fisher's exact test. The p value was corrected by multiplication by (N-1) where N is the number of observed alleles. A 95% confidence interval was used to determine possible statistical significance. This statistical analysis was performed as described previously .
Twenty-four patients with POHS were typed for the alanine repeat polymorphism present in the TM region of the MICA gene. One hundred and six controls were also typed for the alanine repeat polymorphism. Five of the possible seven MICA alleles, based on the alanine repeat polymorphism, were found in the patient and in the control group. The MICA-A7 and MICA-A10 allele were not found in either group.
Table 1 shows the MICA typing results for the POHS patients with POHS scores=1 or 2 (group 2) and POHS score=3 (group 1) in this study. The MICA genotype frequencies for the 24 patients, in the complete and the subdivided groups, and the 106 controls are listed in Table 2. No statistically significant differences were found in genotype frequencies between controls and patients. Seven patients were homozygous for the MICA alanine repeat polymorphism. Six of them were homozygous for the MICA-A5.1 allele and one patient was homozygous for the MICA-A9 allele. In the control group, a total of 24 homozygous persons was observed. Two persons were homozygous for the A4 allele, 16 for the A5.1 allele, 2 for the A6 allele, and 4 for the A9 allele. The distribution of MICA alleles was in Hardy-Weinberg equilibrium in both groups. Homozygosity analysis of each MICA allele did not reveal any significant differences between the patient and the control group (data not shown).
To determine whether an association of POHS with the MICA alanine repeat polymorphism is present in either the total patient group or one of the subdivided patient groups, phenotype frequencies of the MICA alleles in the total and subdivided patient groups and the control group were compared. Table 3 shows the phenotype frequencies of the 5 different MICA alleles in the total patient group, the subdivided patient group 1 (POHS score=3), the subdivided patient group 2 (POHS scores=1 or 2), and the control group. The phenotype frequencies found in the control group are in concordance with previously reported phenotype frequencies for the Caucasian population . At phenotypic level, no statistically significant differences were observed in frequency between the complete patient group and the control group. The MICA-A5.1 allele was the most frequent allele in the patient and in the control group with respectively 58.3% and 62.3%. The lowest phenotype frequency was found for the MICA-A4 allele in both patient and control group, respectively 16.6% and 15.1%. After subdividing the total patient group in group 1 (POHS score=3) and group 2 (POHS scores=1 or 2), also no statistically significant differences were observed compared with the control group; however the number of patients becomes quite low in the subdivided patient groups.
In this study we investigated whether an association exists between the alanine repeat polymorphism in the MICA TM region and POHS. POHS patients and controls were evaluated for their alanine repeat polymorphism. Our results show that no statistical significant differences were observed for one of the MICA alleles based on the alanine repeat. Neither between the total patient group and the control group, nor between the control group and either of the subdivided patient groups, group1 with POHS score 3 and group 2 with POHS score 1 or 2.
POHS was in the past thought to be caused by an infectious agent, Histoplasma capsulatum, but this correlation has never been proven. In patients from the United States an association with the HLA-B7-DR2 haplotype was observed in the past [5,6]. A recent study has shown a strong association with HLA-DR15 (DR2) in Dutch patients, with in the same study identifying a critical amino acid at the peptide binding site within the HLA-DR molecule: isoleucine at position 67 . Since linkage disequilibrium exists between B and DR, the gene involved might be any gene in this region. The stress-inducible cell surface molecule MICA is one of the HLA related genes, located close to HLA-B and has shown to be associated as a primary or secondary genetic marker in several diseases previously thought to be caused by a HLA class I or II association [10,11,14,15]. In this study MICA was thought to be a good candidate gene. Dutch POHS patients are not infected with Histoplasma capsulatum , indicating that also no peptide of this infectious agent could be presented to elicit an immune response. However, for stable MICA expression and for eliciting an immune response via NK cells, no peptide-presentation by the MICA molecule and a beta-2-microglobuline chain is required as is the case for HLA molecules.
From our study, we can conclude that the MICA transmembrane polymorphism is not associated with the disease POHS. In the B7-DR2 associated haplotype, DR has shown to be crucial in POHS at amino acid position 67 . The linkage disequilibrium between MICA and HLA-B is stronger than between MICA and HLA-DR explaining the lack of association with MICA. Our results confirm the earlier described linkage disequilibrium between HLA-B and MICA (data not shown) [17,18]. However, within the B-DR region, genes have been identified in the Human Genome Project  that might be potential candidate genes associated with POHS.
The number of patients was limited in this study; however, the described association with B7-DR2 was confirmed in this patient panel . It is interesting to study the associations found in this and earlier studies  in POHS patients from endemic and non-endemic regions in the United States. The heterogeneous classification in different ethnic groups should be standardized according to uniform criteria . Heterogeneity within the POHS groups requires many patient samples collected from many ophthalmic centres worldwide to define candidate genes in the HLA-B-DR region.
This study was supported with a grant from the "Algemene Nederlandse Vereniging ter Voorkoming van Blindheid". The authors acknowledge the collaboration with Dr. H. Otten for the use of the control samples.
1. Gass JD, Wilkinson CP. Follow-up study of presumed ocular histoplasmosis. Trans Am Acad Ophthalmol Otolaryngol 1972; 76:672-94.
2. Ongkosuwito JV, Kortbeek LM, Van der Lelij A, Molicka E, Kijlstra A, de Smet MD, Suttorp-Schulten MS. Aetiological study of the presumed ocular histoplasmosis syndrome in the Netherlands. Br J Ophthalmol 1999; 83:535-9.
3. Ongkosuwito JV, Tilanus MG, Van der Lelij A, van Schooneveld MJ, Jager MJ, Rozemuller EH, de Smet MD, Suttorp-Schulten MS. Amino acid residue 67 (isoleucine) of HLA-DRB is associated with POHS. Invest Ophthalmol Vis Sci 2002; 43:1725-9.
4. Smith RE, Ganley JP. An epidemiologic study of presumed ocular histoplasmosis. Trans Am Acad Ophthalmol Otolaryngol 1971; 75:994-1005.
5. Meredith TA, Smith RE, Braley RE, Witkowski JA, Koethe SM. The prevalence of HLA-B7 in presumed ocular histoplasmosis in patients with peripheral atrophic scars. Am J Ophthalmol 1978; 86:325-8.
6. Meredith TA, Smith RE, Duquesnoy RJ. Association of HLA-DRw2 antigen with presumed ocular histoplasmosis. Am J Ophthalmol 1980; 89:70-6.
7. Klein J, Sato A. The HLA system. First of two parts. N Engl J Med 2000; 343:702-9.
8. Complete sequence and gene map of a human major histocompatibility complex. The MHC sequencing consortium. Nature 1999; 401:921-3.
9. Shiina T, Tamiya G, Oka A, Takishima N, Yamagata T, Kikkawa E, Iwata K, Tomizawa M, Okuaki N, Kuwano Y, Watanabe K, Fukuzumi Y, Itakura S, Sugawara C, Ono A, Yamazaki M, Tashiro H, Ando A, Ikemura T, Soeda E, Kimura M, Bahram S, Inoko H. Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region. Proc Natl Acad Sci U S A 1999; 96:13282-7.
10. Goto K, Ota M, Ohno S, Mizuki N, Ando H, Katsuyama Y, Maksymowych WP, Kimura M, Bahram S, Inoko H. MICA gene and ankylosing spondylitis: linkage analysis via a transmembrane-encoded triplet repeat polymorphism. Tissue Antigens 1997; 49:503-7.
11. Mizuki N, Ota M, Kimura M, Ohno S, Ando H, Katsuyama Y, Yamazaki M, Watanabe K, Goto K, Nakamura S, Bahram S, Inoko H. Triplet repeat polymorphism in the transmembrane region of the MICA gene: a strong association of six GCT repetitions with Behcet disease. Proc Natl Acad Sci U S A 1997; 94:1298-303.
12. Wallace GR, Verity DH, Delamaine LJ, Ohno S, Inoko H, Ota M, Mizuki N, Yabuki K, Kondiatis E, Stephens HA, Madanat W, Kanawati CA, Stanford MR, Vaughan RW. MIC-A allele profiles and HLA class I associations in Behcet's disease. Immunogenetics 1999; 49:613-7.
13. Yabuki K, Mizuki N, Ota M, Katsuyama Y, Palimeris G, Stavropoulos C, Koumantaki Y, Spyropoulou M, Giziaki E, Kaklamani V, Kaklamani E, Inoko H, Ohno S. Association of MICA gene and HLA-B*5101 with Behcet's disease in Greece. Invest Ophthalmol Vis Sci 1999; 40:1921-6.
14. Sugimura K, Ota M, Matsuzawa J, Katsuyama Y, Ishizuka K, Mochizuki T, Mizuki N, Seki SS, Honma T, Inoko H, Asakura H. A close relationship of triplet repeat polymorphism in MHC class I chain-related gene A (MICA) to the disease susceptibility and behavior in ulcerative colitis. Tissue Antigens 2001; 57:9-14.
15. Choi HB, Han H, Youn JI, Kim TY, Kim TG. MICA 5.1 allele is a susceptibility marker for psoriasis in the Korean population. Tissue Antigens 2000; 56:548-50.
16. Gonzalez S, Martinez-Borra J, Torre-Alonso JC, Gonzalez-Roces S, Sanchez del Rio J, Rodriguez Perez A, Brautbar C, Lopez-Larrea C. The MICA-A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to the development of psoriatic arthritis and is independent of the association of Cw*0602 in psoriasis. Arthritis Rheum 1999; 42:1010-6.
17. Yao Z, Volgger A, Helmberg W, Keller E, Fan LA, Chandanayingyong D, Albert ED. Definition of new alleles of MIC-A using sequencing-based typing. Eur J Immunogenet 1999; 26:225-32.
18. Tian W, Boggs DA, Ding WZ, Chen DF, Fraser PA. MICA genetic polymorphism and linkage disequilibrium with HLA-B in 29 African-American families. Immunogenetics 2001; 53:724-8.
19. Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science 1998; 279:1737-40.
20. Stephens HA. MICA and MICB genes: can the enigma of their polymorphism be resolved? Trends Immunol 2001; 22:378-85.
21. Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 1999; 285:727-9.
22. Jinushi M, Takehara T, Kanto T, Tatsumi T, Groh V, Spies T, Miyagi T, Suzuki T, Sasaki Y, Hayashi N. Critical role of MHC class I-related chain A and B expression on IFN-alpha-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection. J Immunol 2003; 170:1249-56.
23. Rueda B, Pascual M, Lopez-Nevot MA, Gonzalez E, Martin J. A new allele within the transmembrane region of the human MICA gene with seven GCT repeats. Tissue Antigens 2002; 60:526-8.
24. Bahram S. MIC genes: from genetics to biology. Adv Immunol 2000; 76:1-60.
25. Suemizu H, Radosavljevic M, Kimura M, Sadahiro S, Yoshimura S, Bahram S, Inoko H. A basolateral sorting motif in the MICA cytoplasmic tail. Proc Natl Acad Sci U S A 2002; 99:2971-6.
26. Groh V, Bahram S, Bauer S, Herman A, Beauchamp M, Spies T. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc Natl Acad Sci U S A 1996; 93:12445-50.
27. Zwirner NW, Dole K, Stastny P. Differential surface expression of MICA by endothelial cells, fibroblasts, keratinocytes, and monocytes. Hum Immunol 1999; 60:323-30.
28. Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci U S A 1999; 96:6879-84.
29. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16:1215.
30. Svejgaard A, Ryder LP. HLA and disease associations: detecting the strongest association. Tissue Antigens 1994; 43:18-27.