Introduction

Alopecia areata (AA) is an organ-specific autoimmune disease caused by T-cell infiltrates surrounding hair follicles. The AA phenotype can vary from mild, patchy hair loss (AA) to the total loss of scalp hair (alopecia totalis (AT)) or scalp and body hair (alopecia universalis (AU)). This suggests that AA is a multigenic, complex trait, and its discordance in twins suggests that it is triggered by environmental factors such as stress or viral infections in susceptible hosts (Cole and Herzlinger, 1984; Jackow et al., 1998). The prevalence of AA is approximately 0.1–0.2% in the US population, determined by a National Health and Nutrition Examination Survey, 1971–1974 (Safavi, 1992).

HLA, encoded by the major histocompatibility complex on chromosome 6, are probable major susceptibility loci in AA and other autoimmune diseases. In previous studies, HLA-DQB1*03 (*0301–*0303) alleles were present in 80% of all patients with AA, regardless of phenotype, and in 92% of patients with AT or AU (odds ratio=12.14, P=0.00003, corrected). HLA-DRB1*1104 was associated with patchy AA (odds ratio=16) (Welsh et al., 1994; Duvic et al., 1995; Colombe et al., 1999; de Andrade et al., 1999). Whether specific HLA alleles or other HLA region genes, such as tumor necrosis factor (TNF)-, TNF-, Notch, or major histocompatibility complex class I chain-related gene A (MICA), determine susceptibility to AA is not known.

The MICA molecule (previously PERB 11.1 molecule) is a heat-shock- or oxidative-stress-induced antigen (Groh et al., 1996; Yamamoto et al., 2001) belonging to the MIC gene family, and is located about 46.5 kb centromeric to the HLA-B gene. Five MICA polymorphisms (A4, A5, A5.1, A6, and A9), determined by the number of alanine (GCT) repeats, are located in the region encoding the transmembrane domain. The A5.1 allele contains five GCT repeats, plus one extra guanine nucleotide (GCT)₂G(GCT)₂ which causes a frame-shift mutation with a premature termination codon, TAA. The truncated protein, unlike the other MICA alleles, is poor in hydrophobic amino acids, and may encode a soluble, secreted form of MICA (Bahram et al., 1996). In order to further study HLA gene associations in families, we used polymorphic markers overlapping the entire HLA region.

Results

To study the association of AA with the entire HLA locus, 81 individuals from 10 multiplex families, including 38 affected with AA, were genotyped in stage 1 using 10 selected polymorphic markers (described in Materials and methods). Preliminary evidence of association was found only between MICA and AA. However, markers M6S151 and D6S2447 were not informative for this study because the allelic distributions in the family members were not polymorphic (data not shown). In stage 2, we expanded the study by genotyping 176 affected and 231 unaffected members of an additional 84 families for MICA alleles (Table 1). The MICA allele 5.1 (MICA^*5.1) was the most commonly encountered in both affected individuals (40.1%) and unaffected relatives (41.3%) (Table 1). No difference in the frequencies of MICA^*5.1 was found between 94 individuals with patchy AA (38.8%) and 82 individuals with AT/AU (41.5%) (P=0.80).

Table 1 - Distribution of the MICA allele frequencies (%) in 94 multiplex alopecia areata families.

Full table

As shown in Table 2, FBAT analysis was performed on members of 94 families, using the unaffected parents and siblings as controls. No significant association for any AA severity phenotype was found between the MICA locus and AA (P=0.099). However, the MICA allele 6 (MICA^*6) was significantly associated with all collective AA phenotypes (P=0.0083). FBAT analyses were also performed for individual AA phenotypes (patchy vs AT/AU) for each allele, as well as all alleles combined. For the entire MICA locus, there were no statistically significant associations between MICA locus and patchy AA (P=0.155) or the severe AT/AU phenotype (P=0.335). When the individual MICA alleles were examined, the MICA^*5.1 allele showed suggestive evidence of association with patchy AA (P=0.029) (Table 2).

Table 2 - MICA locus and MICA allelic association with AA using FBAT in 94 multiplex families.

Full table

Haplotype analysis results are shown in Tables 3, 4 and 5. To simplify the results of highly polymorphic HLA-DQ alleles, we categorized them as DQ2 (DQB1*0201), DQ7 (DQB1*0301), DQ8 (DQB1*0302), DQ9 (DQB1*0303), DQ4 (all DQB1*0400's alleles), and DQ1 (all DQB1*0500–*0600's alleles) (Marsh et al., 2005). Recent HLA nomenclature published by Marsh et al. (2005) divides previous DQ1 alleles into DQ5 (all DQB1*0500's alleles), DQ6 (most DQB1*0600's alleles), and DQ1 (DQB1*061101, *061102, and *0612's alleles). As we did not separate the DQB1*0500's and DQB1*0600's alleles, we kept the previous nomenclature for those particular alleles. The results show that extended haplotypes DQ1-DR6-MICA^*5.1 (P=0.004) and DQB1*0201-DR3-MICA^*5.1 (P=0.009) are significantly associated with AA. The extended haplotypes DQB1*0201-DR3-MICA^*5.1 (P=0.008) and DQB1*0302-DR4-MICA*4 (P=0.001) are associated with patchy AA. Furthermore, the extended haplotypes DQ1-DR6-MICA^*5.1 (P=0.037) and DQB1*0301-DR4-MICA*9 (P=0.007) are associated with the most severe phenotype, AT/AU (Tables 3, 4 and 5).

Table 3 - Haplotype analysis of HLA-DR, -DQ, and -MICA in affected AA individuals compared to unaffected related individuals.

Full table

Table 4 - Haplotype analysis of HLA-DR, -DQ, and MICA in patchy AA-affected individuals compared to unaffected related individuals.

Full table

Table 5 - Haplotype analysis of HLA-DR, -DQ, and -MICA in AT/AU-affected individuals compared to unaffected related individuals.

Full table

Discussion

Previous studies showed significant associations between AA and class II HLA-DR and –DQ, but whether those alleles or nearby genes confer disease susceptibility is uncertain (Welsh et al., 1994; Colombe et al., 1999). We previously reported a significant association between three alleles of HLA-DQB1 (*0302, *0601, and *0603) and two alleles of HLA-DR (DR4 and DR6) and AA multiplex families (de Andrade et al., 1999). In this study, in addition to HLA-DR and -DQ, we genotyped same families for MICA.

We show, for the first time, a significant association between the MICA locus and AA, suggested in 10 multiplex families and confirmed in 84 families. Although MICA^*5.1 allele, reported at a frequency of 53% in American Caucasians (Petersdorf et al., 1999), was also the most common allele in our American Caucasians population (40%), MICA^*6 was found to be associated with AA of all severity phenotypes considered together, whereas MICA^*5.1 was associated with patchy persistent AA.

Like HLA alleles, MICA allelic polymorphisms are associated with several autoimmune diseases and vary among different population groups (Ota et al., 1997; Bahram, 2000). In Korean and Japanese populations, MICA^*4 is associated with type 1 diabetes and MICA^*6 is a protective allele (Kawabata et al., 2000; Park et al., 2001). In American Caucasians, diabetes mellitus has been suggested to protect against AA (Wang et al., 1994), and we found that AA is associated with MICA^*6. MICA^*5.1 was associated with mild, patchy AA in this study, and was associated with psoriasis vulgaris in Korean and Chinese populations (Cheng et al., 2000; Choi et al., 2000), and with adult onset of type I diabetes mellitus in Italians (Gambelunghe et al., 2001). Ethnicity may determine specific MICA allelic associations with autoimmune diseases.

MICA is a candidate gene for susceptibility to autoimmune diseases, including AA. MICA is a ligand for a heterodimer receptor NKG2D-DAP10 found on natural killer cells, T cells, and CD8+ T cells (Bauer et al., 1999), and is expressed on the surface of epithelial cells, endothelial cells, fibroblasts, keratinocytes, and monocytes (Zwirner et al., 1999). MICA is also present in the epidermis of normal and psoriasis lesions and hair follicles, where its presence could attract cytotoxic or natural killer cells bearing its receptor (Tay et al., 2000). The MICA^*5.1 found in association with patchy AA encodes a truncated protein transported in the apical, rather than basolateral, membrane of the epithelial cells (Bahram, 2000; Suemizu et al., 2002).

To determine whether a region, rather than genes, is associated with AA, we performed extended haplotype analysis for the susceptibility genes HLA-DQ, -DR, and MICA. Extended haplotypes DQB1*0201-DR3-MICA^*5.1 (P=0.008) and DQB1*0302-DR4-MICA*4 (P=0.001) were significantly associated with patchy AA, while extended haplotypes DQ1-DR6-MICA^*5.1 (P=0.037) and DQB1*0301-DR4-MICA*9 (P=0.007) were significantly associated with severe AA. Considering all phenotypic severity together, extended haplotypes DQ1-DR6-MICA^*5.1 (P=0.004) and DQB1*0201-DR3-MICA^*5.1 (P=0.009) were significantly associated with AA. This further supports our previous finding of a highly significant association between -DQB1*03 alleles (*0301–0303) and AT/AU in unrelated individuals and in identical twins (Welsh et al., 1994; Jackow et al., 1998). The extended haplotype association may confer increased susceptibility of the locus to AA, and may distinguish the mild from the severe phenotype. Although MICA is in linkage disequilibrium with HLA-DQ and -DR (Kawabata et al., 2000; Bilbao et al., 2002), MICA^*6 could also confer susceptibility to AA. To understand the role of MICA in AA, further association studies with unrelated controls and functional studies of the MICA^*5.1 and ^*6 alleles are needed.

Materials and Methods

Data collection

Study participants were identified in University of Texas dermatology clinics and through a website: http://www.mdanderson.org/departments/alopecia. Eligibility criteria for probands as well as involved family members included a diagnosis and phenotype determination of AA by the proband and the relative's local dermatologists. The criteria used were patchy AA (if there was any patchy hair loss on the scalp or body), AT (complete scalp hair loss), or AU (complete scalp and body hair loss). The study was conducted according to the Declaration of Helsinki principles. Participants gave written informed consent approved by the institution review board to have blood samples drawn for genotyping. DNA was extracted from fresh peripheral whole blood using standard methods (Stratagene, La Jolla, CA).

Polymerase chain reaction and sequencing analysis

Genomic DNA (20–100 ng) was used to genotype by polymerase chain reaction. MICA and TNF- genes plus eight microsatellite markers (M6S232, M6S233, D6S2447, M6S115, M6S234, M6S166, M6S101, and M6S151) previously described by Nair et al. (2000) at website http://www.psoriasis.umcih.edu/hla2000/index.html were used to genotype the HLA region in 10 multiplex families initially. Polymerase chain reaction conditions for all polymorphic markers were the same as those recommended by Nair et al., with the exception of the M6S232, M6S233, and M6S234 markers, which were cooled at 65°C for 1 minute. Polymerase chain reaction products were sized on a 2.5% ethidium bromide agarose gel with "BioMarker™ Low" DNA ladder (BioVentures Inc., Murfreesboro, TN), followed by DNA sequencing gels run on ABI 3100 and AB 3730XL Genetic Analyzers (Applied Biosystems, Foster City, CA).

Statistical analysis

To investigate the genetic composition of the major histocompatibility complex region, we used a two-stage approach to select the genetic markers. Under this approach, all the markers are genotyped and tested at stage 1 using a subset of affected cases and unaffected relatives, and the most promising markers are genotyped on the remaining individuals and tested using all the individuals at stage 2. This two-stage approach was shown to reduce the cost of the study by half, with enough power to detect promising genetic markers (Satagopan and Elston, 2003; Satagopan et al., 2004). In stage 1, eight markers (M6S232, M6S233, D6S2447, M6S115, M6S234, M6S166, M6S101, and M6S151) and two genes (TNF- and MICA) were genotyped in 10 of the largest multiplex American Caucasian families (81 total individuals, of whom 38 had AA). In stage 2, only the significant genetic markers were genotyped in the other 84 families.

The family-based association tests (FBAT) (Laird et al., 2000; Rabinowitz and Laird, 2000) were applied to identify the significant genetic markers using the program FBAT (http://www.biostat.harvard.edu/~fbat/fbat.htm). FBAT belongs to a class of tests that utilize within- and between-family marker-inheritance patterns to test for association and therefore are protected, by design, from the confounding effect caused by an admixture. Different types of designs, from sibships to extended families, can be used in the FBAT program. FBAT evaluates the association between the specific genetic marker and AA, and also the allelic association in a specific marker and AA.

A genetic marker was considered to be genotyped in stage 2 if it showed suggestive association (P-value between 0.05 and 0.01). In stage 2, we reported only results that were statistically significant (P-value between 0.01 and 0.001) and highly significant (P-value <0.001). The multiple comparisons take into consideration these lower P-values. The frequency of each allele was expressed as a percentage of the total number of alleles. The frequencies were estimated from the pedigree data using the method proposed by Boehnke (1991) and the ILINK program of the LINKAGE package (Lathrop et al., 1984).

In order to conduct a genetic dissection of the HLA region that will help to identify the causative variants, we performed a haplotype analysis including MICA, HLA-DR, and HLA-DQ alleles using 176 affected and 231 unaffected related individuals (Lange et al., 2003). We used the method developed by Horvath et al. (2004), which is implemented in the software FBAT.

Conflict of Interest

The author states no conflict of interest.

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Acknowledgments

This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin (NIAMS) Grant AR45789, K24-CA86815, the National Alopecia Areata Foundation (NAAF) research grant, and the grant from Zarrow Family Foundation. This work was presented at the May 2004 meeting of the Society of Investigative Dermatology, in Providence, RI. We thank The University of Texas MD Anderson Cancer Center's core-laboratory facility, funded by NCI Grant CA-16672 (DNA Analysis Core Facility), for performing tests with the ABI 3100 and AB 3730XL Genetic Analyzer.