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MICA genetic polymorphism and HLA-A,C,B,MICA and DRB1 haplotypic variation in a southern Chinese Han population: Identification of two new MICA alleles, MICA*060 and MICA*062
Human Immunology 72 (2011) 510-515
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MICA genetic polymorphism and HLA-A,C,B,MICA and DRB1 haplotypic variation in a southern Chinese Han population: Identification of two new MICA alleles, MICA*060 and MICA*062 Wei Tian*, JinHong Cai, XueXiang Liu Immunogenetics Research Group, Department of Immunology, College of Basic Medical Sciences, Central South University, Changsha, Hunan, P. R. China
A R T I C L E
I N F O
Article history: Received 12 January 2011 Accepted 31 March 2011 Available online 8 April 2011
Keywords: HLA MICA Sequence-based typing Linkage disequilibrium Southern Chinese Han population
A B S T R A C T
In this study, 201 healthy, unrelated Han subjects in Hunan province, southern China, were investigated by sequence-based typing (SBT) for the allelic variation of the human major histocompatibility complex (MHC) class I chain-related gene A (MICA). Nineteen MICA alleles were observed, among which MICA*008:01 predominated with gene frequency of 30.35%. There was significant linkage disequilibrium (LD) of MICA*012:01 with HLA-B*54 and HLA-B*55, which was not observed in a northern Chinese Han population. Haplotype HLA-A*11-C*07-B60-MICA*008:01 (9.16%) was highly specific to this southern Chinese Han population. The most common five-locus haplotype in this population was HLA-A*02-C*01-B*46-MICA*010DRB1*09 (8.73%). A new MICA allele, MICA*060, was identified on an HLA-A*02-C*01-B*55:02-DRB1*14 haplotype through extended family analysis. MICA*060 has probably arisen from MICA*012:01. Another new MICA allele, MICA*062, was identified by screening 1432 subjects using polymerase chain reaction– sequence-specific priming technology. MICA*062 has probably derived from MICA*010. Of particular interest is that MICA*062 was carried on an HLA-C*08-B*48:01-DRB1*14 haplotypic segment, as HLA-B*48 has been consistently shown to be primarily linked to MICA gene deletion in east Asian populations. Our results provide new insight into MICA genetic polymorphism in human populations. The findings reported here are of importance for future studies on the potential role of MICA in allogeneic organ transplantation and disease association in populations of Chinese ancestry. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The human major histocompatibility complex (MHC) class I chain-related gene A (MICA) is located 46 kb centromeric to human leukocyte antigen gene B (HLA-B); it is composed of six exons that encode the leader peptide, three extracellular domains (␣1, ␣2, and ␣3), a transmembrane segment, and a carboxy-terminal cytoplasmic tail, respectively [1]. Unlike classical HLA molecules, MICA does not associate with -2-microglobulin and peptides derived from antigen processing but acts as a ligand for the activating NKG2D receptor mainly expressed by ␥␦ T cells, CD8⫹ ␣T cells, and natural killer (NK) cells, facilitating the recognition and elimination of virus-infected or transformed cells by the host immune system [2]. In contrast to other nonclassical HLA genes, MICA gene exhibits remarkable allelic diversity [3], MICA polymorphisms have been shown to be associated with a number of pathophysiological disorders, including malignancy, pathogen infection, autoimmune disease, and allograft rejection or graft-versus-host disease after allogeneic transplantation [4 –9].
* Corresponding author. E-mail address: [email protected] (W. Tian).
The Han Chinese represents about 94% of the population in China and could be categorized into Northern and Southern subgroups, which are separated approximately by the Yangtze River [10]. Hunan province is situated in the south of the middle Yangtze river valley and has a population of about 64 million (2000 census), in which Han ethnic group constitutes the largest ethnic group and comprises about 90% (⬇58 million) of the population in this region. There are also minority groups in this region, including Tujia, Miao, Dong, Yao, Hui, and others. According to the historical records, Hunan was inhabited by the ancestors of the modern Miao, Tujia, Dong, and Yao peoples until around 350 BC, when migration of Han Chinese from the central and north China occurred. Such migration continued for hundreds of years thereafter because of war or drought in the north. Currently, information on MICA alleles and MICA-containing HLA haplotypes in Chinese Han populations residing in different geographic locations is still limited. In a recent study, MICA allelic variation was investigated in a northern Chinese Han population [11]. In the present study, we investigated MICA allelic distribution in a Chinese Han population in Hunan province, in which a novel MICA allele, MICA*060, was identified. MICA*060 has an adenine substitution at position 862 in exon 4, which has only been recently
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.03.006
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reported for MICA*058 (http://hla.alleles.org/data/txt/mica_nuc. txt). As an extension of our study, we sought to determine the distribution of this adenine replacement in more diverse populations by screening additional 1432 samples. Unexpectedly, another novel MICA allele, MICA*062, was observed.
2.2. Extraction of genomic DNA from peripheral blood
2. Subjects and methods
A comprehensive strategy was used for MICA typing, as recently described by us [11]. Briefly, polymerase chain reaction-sequencebased typing (PCR-SBT) was used for the analysis of MICA exons 2, 3, and 4, fluorescent polymerase chain reaction–fragment analysis was used to determine the (GCT)n microsatellite at exon 5 of MICA gene (abbreviated as MICA-STR) [4,11], and polymerase chain reaction–sequence-specific priming (PCR-SSP) was applied to detect MICA gene deletion (MICA*Del) [4,11]. MICA allele assignment was based on the integrated information of exons 2–5.
2.1. Subjects The sample panel used for MICA population genetics consisted of 201 healthy, unrelated individuals of Han ethnicity. Among them, the subject carrying MICA*060 was a 44-year-old woman (sample ID: W124) (Fig. 1) living in northern China, whose maiden family had resided for the last three generations in Hunan province; the other 200 individuals were native residents in Hunan province. Among the 201 samples were 46 parental samples derived from 23 nuclear families. This sample panel comprised 59.2% men (119/ 201) and 40.8% women (82/201). The second sample panel comprised 1432 healthy, unrelated individuals, which were used to screen for the adenine substitution at position 862 in exon 4 (http://hla.alleles.org/data/txt/mica_nuc. txt). Among them were 1299 subjects of Han ethnicity in Hunan province, 67 subjects of ethnic minority (Hui, Miao, Tujia, Dong and Yao) in Hunan province, and 66 subjects of Han ethnicity in central and southeastern China. This sample panel consisted of 59.8% men (857/1432) and 40.2% women (575/1432). Ethnicity and ancestry was reported by each participant.A blood sample was taken with each person’s informed consent. There were no missing genotypic data. All protocols were approved by the Institutional Review Committee of local authorities.
Genomic DNA was extracted from 2 to 5 ml peripheral blood using a standard salting-out protocol [4]. 2.3. MICA typing
2.4. Screening of adenine substitution at position 862 in exon 4 A pair of sequence-specific PCR primers were designed to detect the adenine substitution at position 862 in exon 4 (http://hla.alleles. org/data/txt/mica_nuc.txt). The sequences of sense primer and antisense primer are 5=-ACATGGAACACAGCA-3= and 5=-ATGACTCTGAAGCAC-3=, respectively. The sense primer corresponds to the genomic segment from nucleotide position 8588 – 8602, and the antisense primer is complementary to genomic segment from nucleotide position 8743– 8757, when aligned with MICA consensus sequence available under accession number X92841. This pair of primers generate a 170-bp product when adenine substitution at position 862 in exon 4 is present. A pair of PCR primers were used as internal positive control, the primer sequences are 5=-TGCCAAGTGGAGCACCCAA-3= and 5=-GCATCTTGCTCTGTGCAGAT-3=, respec-
Fig. 1. Family segregation analysis of MICA*060. Haplotype is expressed in order of HLA-A-C-B-MICA-DRB1(from top to bottom). Samples W119 and W120 were typed only for HLA-A, C, B, and MICA loci; samples S103 and S104 were typed only for MICA locus. Samples W118, W119, and W120 were northern Chinese Hans: samples S102, S103, S104, and S105 were southern Chinese Hans residing in Hunan province; the proband, W124, currently resides in northern China.
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tively. This pair of primers amplify a 796-bp fragment from the third intron of HLA-DRB1 [12]. PCR amplification was carried out in a 10-l volume, containing 1X PCR buffer, 1.5 mmol/l MgCl2, 0.2 mmol/l dNTPs, 2.5 pmol of each SSP primer, 0.3 pmol of each internal control primer, 0.8 U of Taq polymerase (Promega, Shanghai, China), and 50⬃100 ng genomic DNA. A blank control, for which double-distilled water was used as DNA template, was included in each batch of PCR reactions. Amplification was performed using a MyCycler (Bio-Rad, Hercules, CA), cycling temperature profiles were as follows: the reaction mixture was subjected to one cycle of denaturation at 95⬚C for 2 minutes followed immediately by 30 cycles of 95⬚C for 20 seconds, 61⬚C for 60 seconds, and 72⬚C for 60 seconds, and a final extension at 72⬚C for 5 minutes before cooling to 15⬚C. PCR amplicons were identified by running 5 l of PCR product on a 2% agarose gel. The image of each gel was obtained using a bioimaging system (UVP, Upland, CA), and the size of the amplicons was determined by comparison against the migration of a 100-bp DNA ladder. 2.5. Cloning of exon 4 of 2 novel MICA alleles During MICA-sequence-based typing (SBT) of the 201 samples, sample W124 showed a novel sequence in exon 4, which differed from MICA*012:01 by an adenine substitution at position 862 or, alternatively, differed from MICA*002:01 by an adenine substitution at this position. To verify this polymorphism, PCR product of MICA exon 4 derived from sample W124 was therefore cloned using a TA cloning kit (TaKaRa, Dalian, China) [11]. The inserts of 20 randomly selected recombinant plasmids were sequenced in both directions. Two of the 1432 subjects, both of which were of Han ethnicity in Hunan province, were found to carry the adenine substitution at position 862 in exon 4. Interestingly, PCR-SBT indicated the existence of another new MICA allele, which was related to MICA*010. PCR products of MICA exon 4 derived from both samples (sample IDs: T156 and T450, respectively) were therefore cloned, and the inserts of 10 randomly selected recombinant plasmids from each sample were sequenced in both directions. 2.6. HLA typing HLA-A, C, and B data by generic PCR-SSP have been reported for the majority of the 201 samples [13,14]. In the present study, HLA-DRB1 together with HLA-A, C, and B loci of the remaining samples were typed by PCR-SSP using commercial typing reagents (Genovision, Westchester, PA). Subtyping of HLA-B*13, selected subtyping of HLA-B*48 and HLA-B*55 associated with the novel MICA alleles was accomplished by PCR-SSP using commercial subtyping kits (Invitrogen, Brown Deer, WI; Genovision, Westchester, PA). 2.7. Statistical analysis The program ARLEQUIN 3.11 (http://cmpg.unibe.ch/software/ arlequin3/) [15] was used to analyze the data, including the Hardy– Weinberg equilibrium (HWE) test, multilocus haplotype inference, and pairwise global linkage disequilibrium (LD). Maximum likelihood haplotype frequencies were estimated using the expectationmaximization (EM) algorithm, as implemented in the program ARLEQUIN 3.11. The classic coefficients of LD, ⌬, and of normalized LD, ⌬rel, were computed for each individual haplotype as described previously [11,14]. Fisher exact test was performed to determine the significance of ⌬rel. The LD analysis was restricted to HLA-BMICA haplotypes with observed frequencies ⬎1.5%. Statistical significance was defined at the 5% level after the p value was adjusted using Bonferroni’s correction, that is, by multiplying the p value by the number of independent comparisons performed.
3. Results 3.1. Hardy–Weinberg equilibrium test of MICA locus The MICA genotypic distributions were consistent with Hardy– Weinberg proportions (p ⫽ 0.1602). 3.2. MICA allele frequencies The MICA allele frequencies are summarized in Table 1. Nineteen MICA alleles were detected in this population, among which MICA*008:01 predominated with gene frequency of 30.35%. MICA*054, recently reported in a Caucasian individual [16], was observed in this population and associated with (GCT) 5 in exon 5. MICA*059, recently identified in a northern Chinese Han population [11], was also detected in this population. 3.3. LD between HLA-B and MICA All 13 kinds of HLA-B-MICA haplotypes with a frequency ⬎1.5% were in significant LD, among which 5 haplotypes showed a normalized LD value of 1 (Table 2). HLA-B*13:01 and HLA-B*13:02 displayed completely different LD patterns with MICA gene; conversely, multiple HLA-B lineages were also found in tight LD with the same MICA allele, for example, HLA-B*35, HLA-B*38 and HLAB*58 were all in strong LD with MICA*002:01. 3.4. HLA-A-C-B-MICA haplotypic diversity There were 23 kinds of HLA-A-C-B-MICA haplotypes occurring at least four times (with a frequency of ⱖ1%) (Table 3). Haplotypes HLA-A*02-C*01-B*46-MICA*010, HLA-A*11-C*07-B60-MICA*008: 01, and HLA-A*02-C*07-B60-MICA*008:01 were most prevalent, with frequencies of 14.5%, 9.16%, and 4.09%, respectively. Some haplotypes appeared to have derived from a common ancestral haplotypic block; for example, there were at least five kinds of HLA-B60-MICA*008:01 haplotypes with different HLA-A or HLA-C configurations. 3.5. HLA-A-C–B-MICA-DRB1 haplotypic diversity There were 26 kinds of HLA-A-C-B-MICA-DRB1 haplotypes occurring at least four times (with a frequency of ⱖ1%) (Table 4). HLA-A*02-C*01-B*46-MICA*010-DRB1*09, HLA-A*11-C*07-B60-
Table 1 MICA allele frequencies in 201 southern Chinese Han subjects MICA allelea
Frequency
*002:01 *004 *008:01 *008:02 *009:01 *009:02 *010 *011 *012:01 *017 *019 *020 *027 *033 *045 *054 *059 *Del *060
0.0895 0.0099 0.3035 0.005 0.0547 0.005 0.2462 0.0025 0.0796 0.005 0.087 0.0025 0.0274 0.0025 0.0672 0.0025 0.005 0.0025 0.0025
a
Using the current sequence-based typing (SBT) protocol, MICA *008:04 could not be distinguished from MICA *008:01; these two alleles differ at nucleotide position 21 in exon 1; MICA *049 could not be distinguished from MICA *009:01, these two alleles differ at nucleotide position 1067 in exon 6; MICA *048 could not be distinguished from MICA *027, these two alleles differ at nucleotide position 1017 in exon 5 (http://hla.alleles.org/data/txt/mica_nuc.txt).
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Table 4 Twenty-six kinds of HLA-A-C-B-MICA-DRB1 haplotypes
Table 2 Two-locus linkage disequilibrium analysis between HLA-B and MICA Haplotype
HF
⌬
⌬rel
pa
Haplotypea,b
Number
Frequency
B60-MICA*008:01 B*46-MICA*010 B*13:01-MICA*045 B75-MICA*019 B62-MICA*010 B*51-MICA*009:01 B*55-MICA*012:01 B*58-MICA*002:01 B*54-MICA*012:01 B*13:02-MICA*008:01 B*35-MICA*002:01 B61-MICA*027 B*38-MICA*002:01
0.2537 0.1815 0.0672 0.0521 0.0446 0.0398 0.0348 0.0348 0.0299 0.0274 0.0249 0.0224 0.0199
0.1759 0.1356 0.0622 0.0465 0.0299 0.0375 0.0314 0.0315 0.0273 0.0191 0.0227 0.0218 0.0181
0.9859 0.964 1 0.787 0.6644 0.938 0.8076 0.926 0.9181 1 1 1 1
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0037 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0018 ⬍0.0001 ⬍0.0001 ⬍0.0001
*02-*01-*46-*010-*09 *11-*07-60-*008:01-*12 *02-*01-*46-*010-*08 *33-*03-*58-*002:01-*03 *11-*07-60-*008:01-*08 *11-*08-75-*019-*12 *30-*06-*13:02-*008:01-*07 *02-*07-60-*008:01-*08 *11-*07-60-*008:01-*15 *11-*04-62-*010-*04 *02-*07-60-*008:01-*04 *02-*03-*13:01-*045-*12 *24-*01-*54-*012:01-*04 *11-*07-60-*008:01-*11 *11-*03-60-*008:01-*09 *24-*08-75-*019-*12 *02-*07-60-*008:01-*14 *11-*08-75-*019-*15 *02-*01-*46-*010-*11 *02-*07-*38-*002:01-*08 *11-*01-*55-*012:01-*04 *11-*01-*55-*012:01-*09 *11-*03-*13:01-*045-*12 *24-*03-*13:01-*045-*15 *24-*07-60-*008:01-*09 *11-*03-60-*008:01-*14
35 15 13 9 8 8 8 6 6 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4
0.0875 0.0371 0.0334 0.0224 0.0206 0.0202 0.0199 0.0155 0.0136 0.0133 0.0124 0.0124 0.0119 0.0115 0.0112 0.0111 0.011 0.0103 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.0098
All 13 HLA-B-MICA haplotypes (with a frequency ⬎1.5%) showed values of p ⬍0.00385 (0.05 after Bonferroni’s correction) and were considered to be in significant linkage disequilibrium.
a
MICA*008:01-DRB1*12 and HLA-A*02-C*01-B*46-MICA*010DRB1*08 were the most common haplotypes with frequencies of 8.75%, 3.71%, and 3.34%, respectively. Diversification at HLA-DRB1 locus was found in several common HLA-A-C-B-MICA haplotypes, for example, HLA-A*02-C*01-B*46-MICA*010 was linked to at least three different HLA-DR serologic groups. By contrast, some haplotypes appeared to be conservatively maintained, for instance, haplotype HLA-A*30-C*06-B*13:02-MICA*008:01 was exclusively linked to HLA-DRB1*07.
a
Haplotype is expressed in order of HLA-A-C-B-MICA-DRB1. Only haplotypes occurring at least 4 times (with a frequency of ⱖ1%) are presented.
b
3.6. MICA*060 Among the 20 randomly selected recombinant plasmids, six clones showed a novel sequence (tentatively named MICA*TWFHnew), which when aligned with MICA*012:01, has an adenine substitution at position 862 in exon 4, leading to an amino acid change at codon 265 from Glycine (GGG, MICA*012:01) to Arginine (AGG, MICA*TW-FHnew); six clones had a sequence identical to MICA*012:01; five clones had sequences each differing from MICA*TW-FHnew by one to three mutations at positions other than 862 in exon 4; 3 clones had sequences each differing from MI-
Table 3 Twenty-three kinds of HLA-A-C-B-MICA haplotypes
CA*012:01 by one to two mutations at positions other than 862 in exon 4. Information collected from sequences in exons 2 and 3 suggested a MICA*002/MICA*012 heterozygote. Because MICA* 002:01 and MICA*012:01 share sequence in exon 4, which means that MICA*TW-FHnew could be derived from either of them, family analysis was carried out to resolve the ambiguity. As shown in Fig. 1, MICA*TW-FHnew segregated with sequence motifs of MICA*012:01 on an HLA-A*02-C*01-B*55:02 -DRB1*14 haplotype. The name MICA*060 has been officially assigned to MICA*TWFHnew by the WHO Nomenclature Committee in February 2010. The GenBank accession number for MICA*TW-FHnew is FJ556895. MICA*060 was associated with (GCT) 4 in exon 5.
Haplotypea,b
Number
Frequency
3.7. MICA*062
*02-*01-*46-*010 *11-*07-60-*008:01 *02-*07-60-*008:01 *11-*03-60-*008:01 *11-*08-75-*019 *24-*07-60-*008:01 *11-*03-*13:01-*045 *33-*03-*58-*002:01 *24-*03-60-*008:01 *11-*01-*55-*012:01 *30-*06-*13:02-*008:01 *11-*01-*46-*010 *02-*07-*38-*002:01 *02-*03-*13:01-*045 *11-*01-*54-*012:01 *11-*04-62-*010 *24-*14-*51-*009:01 *24-*01-*46-*010 *02-*15-*51-*009:01 *24-*08-75-*019 *24-*01-*54-*012:01 *24-*03-61-*027 *24-*03-*13:01-*045
58 37 16 14 14 13 13 12 8 8 8 7 7 6 5 5 5 4 4 4 4 4 4
0.145 0.0916 0.0409 0.0352 0.0343 0.0325 0.0323 0.0299 0.0209 0.0199 0.0199 0.018 0.0174 0.0153 0.0136 0.0127 0.0121 0.0111 0.01 0.0098 0.0096 0.0095 0.0094
Analyses of the 10 randomly selected recombinant plasmids derived from PCR product of MICA exon 4 of each sample indicated that this new MICA allele (tentatively named MICA*NEW-SY) had sequence identical to MICA*058 in exon 4. When aligned with MICA*010, MICA*NEW-SY has an adenine substitution at position 862 in exon 4, leading to an amino acid change at codon 265 from glycine (GGG, MICA*010) to arginine (AGG, MICA*NEW-SY). The name MICA*062 has been officially assigned to this new allele by the World Health Organization (WHO) Nomenclature Committee in June 2010. The GenBank accession number for MICA*NEW-SY is HM019306. The genotype of sample T156 was as follows: HLAA*02, A*11; C*01, C*08; B*46, B*48:01; MICA*010, MICA*062; MICA*A5, -; DRB1*11, DRB1*14. The genotype of sample T450 was as follows: HLA-A*02, A*33; C*03, C*08; B*48:01, B*58; MICA*002: 01, MICA*062; MICA*A5, MICA*A9; DRB1*03, DRB1*14. MICA*062 was associated with (GCT) 5 in exon 5. The naming of MICA*060 and MICA*062 follows the agreed-on policy that, subject to the conditions stated in the most recent Nomenclature report [17], names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature report.
a
Haplotype is expressed in order of HLA-A-C-B-MICA. Only haplotypes occurring at least four times (with a frequency of ⱖ1%) are presented. b
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4. Discussion This study was designed to investigate MICA genetic diversity in a southern Chinese Han population by sequence-based typing, for which no such data had previously been available. Our data revealed a high degree of MICA allelic diversification in this population. Similar to most other ethnic groups studied [11,18-21], MICA*008:01 was the most prevalent allele in this population, in contrast to two tribal groups in sub-Saharan Africa [22] and the American Indian populations [23,24], where MICA*002:01, MICA* 020 or MICA*027 predominated. The two most common alleles, MICA*008:01 and MICA*010, combined accounted for about 55% of the MICA gene pool. A large number of MICA alleles were observed only sporadically in this population. HLA-B*13 lineage displayed a clear-cut dichotomy of association with MICA alleles in this southern Chinese Han population, confirming our earlier finding in a northern Chinese Han population [11]. Two individuals in this southern Chinese Han population were found to carry HLA-B*13:01-MICA*059 haplotype. These findings may help to substantiate the notion of southward Chinese Han migration in history. The consistent dichotomous association between HLA-B*13 lineage and MICA alleles is of particular importance to allogeneic transplantation in Chinese Han populations, as evidence is accumulating about the role of MICA in transplant rejection [25]. Subjects with HLA-B*13 may require MICA allelic typing to optimize selection of allogeneic donors. Ten kinds of HLA-B-MICA haplotypes were in significant LD in both the northern and southern Chinese Han populations. However, the distribution of some haplotypes showed a high degree of variability between these 2 groups. For instance, the frequency of haplotype HLA-B60-MICA*008:01 was about sixfold higher in the south (0.2537) than in the north (0.0385) [11]; conversely, the frequency of haplotype HLA-B*13:02-MICA*008:01 decreased southward (0.0962 in the north vs 0.0274 in the south). MICA*012:01, which appeared to be very infrequent in the northern Chinese Han population [11], showed a noticeable frequency in the southern Chinese Han population. Significant LDs of MICA*012:01 with HLA-B*54 and HLA-B*55 were detected in the southern Chinese Han population. Relatively high frequency of MICA*012 has only been reported in very few populations, including Japanese [19], Korean [26], and a population of Jewish descent [21]. LD between MICA*012 and HLA-B*54 or HLA-B*55 was also found in those populations, indicating a common ancestral origin. The fact that MICA*012:01 was found on both HLA-B*54 and B*55 may also reflect mutational origin of the two HLA-B alleles on a single MICA haplotype. A new MICA allele, MICA*060, was observed on an HLA-A*02-Cw*01-B*55:02 -DRB1*14 haplotype in the southern Chinese Han population, suggesting that MICA*060 has probably arisen from MICA*012:01 through a single point mutation. MICA locus displays a G/A dimorphism at position 862 in exon 4 (http://hla.alleles.org/data/txt/mica_nuc.txt). Among the 71 MICA alleles, only MICA*058 and MICA*060 have an adenine at this position. MICA*058, recently described in a Hispanic individual (http:// hla.alleles.org/data/refs/mica_refs.html), was not observed in the southern Chinese Han population. Instead we uncovered another novel MICA allele, MICA*062, which has possibly derived from MICA*010 through a single mutation event. Of particular interest is that MICA*062 was harbored on an HLA-C*08-B*48:01-DRB1*14 haplotypic segment in this southern Chinese Han population, because HLA-B*48 has been consistently shown to be primarily linked to MICA gene deletion (MICA*Del) in east Asian populations [11,19,27]. MICA*054, differing from MICA*010 by a guanine at nucleotide position 871 in exon 4, was also detected in this southern Chinese Han population. All these findings may indicate a relatively rapid evolution of MICA*010 in exon 4.
The inclusion of HLA-C locus helped refine HLA haplotype(s) specific to Chinese sub-populations. For example, HLA-A*11-B60MICA*008:01 was much more common in the southern Chinese Han population (0.1268, Table 3) than in the northern Chinese Han population (0.0192) [11]. A close look revealed that while HLAA*11-C*03-B60-MICA*008:01 showed comparable frequency in these 2 populations (0.0352 vs 0.0192), HLA-A*11-C*07-B60MICA*008:01 was virtually undetectable in the northern Chinese Han population [11], suggesting that this haplotype is highly characteristic of southern Chinese Han population. To our knowledge, this is the first study of its kind to construct HLA-A, C,B, MICA and DRB1 haplotypic repertoire in a single investigation. The multi-locus haplotype data here may provide a unique resource for future studies between human populations. Our study has specific clinical implication in HLA-linked disease association in populations of Chinese ancestry. HLA gene complex has been consistently shown to be associated with nasopharyngeal carcinoma (NPC) [28,29], an epithelial malignancy with the highest incidence rate in southern Chinese populations. Particular HLA-B specificities, mainly including HLA-B*38, B*46 and HLA-B*58 confer high risk for NPC in Chinese populations [30,31]. Evidences are emerging suggesting the involvement of MICA-STR and MICA-129 polymorphism in the susceptibility to NPC [4,32]. Thus, this investigation establishes the extent of linkage disequilibrium between MICA alleles and HLA-B locus, findings shown here are valuable for future haplotype mapping aimed at refining the primary susceptibility locus to NPC, which is most likely located in HLA-B subregion in southern Chinese populations. In conclusion, our data demonstrated a high degree of MICA gene diversity in a southern Chinese Han population. Our study unraveled the peculiarity in terms of MICA allelic and haplotypic repertoire and identified two novel MICA alleles in this population. Findings shown here are of importance for future studies of the potential role of MICA in allogeneic organ transplantation and HLAlinked disease association in populations of Chinese ancestry, and will inform studies of evolution of the MHC gene complex. Acknowledgments We thank all of the blood donors. We also express our gratitude to LiXin Li, Fan Wang, and JunJie Wang for their assistance in the early stage of this project. This work was supported by grants from the Program for New Century Excellent Talents in University (Proj.No. NCET-08-0564), National Natural Science Foundation of China (Proj.No.30671915, 30300311), The Outstanding Youth Science Foundation of Hunan Province (Proj.No.04JJ1007) and Central South University (Proj.No. ZKJ2010039). References [1] Bahram S, Bresnahan M, Geraghty DE, Spies T. A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 1994;91:6259 – 63. [2] Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 1999;285: 727–9. [3] Robinson J, Malik A, Parham P, Bodmer JG, Marsh SG. IMGT/HLA database ⫺ a sequence database for the human major histocompatibility complex. Tissue Antigens 2000;55:280 –7. [4] Tian W, Zeng XM, Li LX, Jin HK, Luo QZ, Wang F, et al. Gender-specific associations between MICA-STR and nasopharyngeal carcinoma in a southern Chinese Han population. Immunogenetics 2006;58:113–21. [5] Kirsten H, Petit-Teixeira E, Scholz M, Hasenclever D, Hantmann H, Heider D, et al. Association of MICA with rheumatoid arthritis independent of known HLADRB1 risk alleles in a family-based and a case control study. Arthritis Res Ther 2009;11:R60. [6] Boukouaci W, Busson M, Peffault de Latour R, Rocha V, Suberbielle C, Bengoufa D, et al. MICA-129 genotype, soluble MICA, and anti-MICA antibodies as biomarkers of chronic graft-versus-host disease. Blood 2009;114:5216 –24. [7] Shojima J, Tanaka G, Keicho N, Tamiya G, Ando S, Oka A, et al. Identification of MICA as a susceptibility gene for pulmonary Mycobacterium avium complex infection. J Infect Dis 2009;199:1707–15.
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[8] Tamaki S, Kawakami M, Yamanaka Y, Shimomura H, Imai Y, Ishida J, et al. Relationship between soluble MICA and the MICA A5.1 homozygous genotype in patients with oral squamous cell carcinoma.Clin Immunol 2009;130:331–7. [9] Viny AD, Clemente MJ, Jasek M, Askar M, Ishwaran H, Nowacki A, Zhang A, et al. MICA polymorphism identified by whole genome array associated with NKG2D-mediated cytotoxicity in T-cell large granular lymphocyte leukemia. Haematologica 2010;95:1713–21. [10] Chen J, Zheng H, Bei JX, Sun L, Jia WH, Li T, et al. Genetic structure of the Han Chinese population revealed by genome-wide SNP variation. Am J Hum Genet 2009;85:775– 85. [11] Tian W, Cai JH, Wang F, Li LX. MICA polymorphism in a northern Chinese Han population: The identification of a new MICA allele, MICA*059. Hum Immunol 2010;71:423–7. [12] Tonks S, Marsh SG, Bunce M, Bodmer JG. Molecular typing for HLA class I using ARMS-PCR: Further developments following the 12th International Histocompatibility Workshop. Tissue Antigens 1999;53:175– 83. [13] Tian W, Li LX, Wang F, Luo QZ, Yan MY, Yu P, et al. MICA-STR, HLA-B Haplotypic diversity and linkage disequilibrium in the Hunan Han population of southern China. Int J Immunogenet 2006;33:241–5. [14] Tian W, Wang F, Cai JH, Li LX. Polymorphic insertions in 5 Alu loci within the major histocompatibility complex class I region and their linkage disequilibria with HLA alleles in four distinct populations in mainland China. Tissue Antigens 2008;72:559 – 67. [15] Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): An integrated software package for population genetics data analysis. Evol Bioinform Online 2005;1: 47–50. [16] Cox ST, Stephens HA, Fernando R, Grant J, Madrigal JA, Little AM. Two novel MICA alleles, MICA*054 and MICA*056. Tissue Antigens 2009;73:85–7. [17] Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, et al. Nomenclature for factors of the HLA system, 2010. Tissue Antigens 2010;75: 291– 455. [18] Petersdorf EW, Shuler KB, Longton GM, Spies T, Hansen JA. Population study of allelic diversity in the human MHC class I-related MIC-A gene. Immunogenetics 1999;49:605–12. [19] Komatsu-Wakui M, Tokunaga K, Ishikawa Y, Kashiwase K, Moriyama S, Tsuchiya N, et al. MIC-A polymorphism in Japanese and a MIC-A-MIC-B null haplotype. Immunogenetics 1999;49:620 – 8.
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Contents lists available at ScienceDirect
MICA genetic polymorphism and HLA-A,C,B,MICA and DRB1 haplotypic variation in a southern Chinese Han population: Identification of two new MICA alleles, MICA*060 and MICA*062 Wei Tian*, JinHong Cai, XueXiang Liu Immunogenetics Research Group, Department of Immunology, College of Basic Medical Sciences, Central South University, Changsha, Hunan, P. R. China
A R T I C L E
I N F O
Article history: Received 12 January 2011 Accepted 31 March 2011 Available online 8 April 2011
Keywords: HLA MICA Sequence-based typing Linkage disequilibrium Southern Chinese Han population
A B S T R A C T
In this study, 201 healthy, unrelated Han subjects in Hunan province, southern China, were investigated by sequence-based typing (SBT) for the allelic variation of the human major histocompatibility complex (MHC) class I chain-related gene A (MICA). Nineteen MICA alleles were observed, among which MICA*008:01 predominated with gene frequency of 30.35%. There was significant linkage disequilibrium (LD) of MICA*012:01 with HLA-B*54 and HLA-B*55, which was not observed in a northern Chinese Han population. Haplotype HLA-A*11-C*07-B60-MICA*008:01 (9.16%) was highly specific to this southern Chinese Han population. The most common five-locus haplotype in this population was HLA-A*02-C*01-B*46-MICA*010DRB1*09 (8.73%). A new MICA allele, MICA*060, was identified on an HLA-A*02-C*01-B*55:02-DRB1*14 haplotype through extended family analysis. MICA*060 has probably arisen from MICA*012:01. Another new MICA allele, MICA*062, was identified by screening 1432 subjects using polymerase chain reaction– sequence-specific priming technology. MICA*062 has probably derived from MICA*010. Of particular interest is that MICA*062 was carried on an HLA-C*08-B*48:01-DRB1*14 haplotypic segment, as HLA-B*48 has been consistently shown to be primarily linked to MICA gene deletion in east Asian populations. Our results provide new insight into MICA genetic polymorphism in human populations. The findings reported here are of importance for future studies on the potential role of MICA in allogeneic organ transplantation and disease association in populations of Chinese ancestry. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The human major histocompatibility complex (MHC) class I chain-related gene A (MICA) is located 46 kb centromeric to human leukocyte antigen gene B (HLA-B); it is composed of six exons that encode the leader peptide, three extracellular domains (␣1, ␣2, and ␣3), a transmembrane segment, and a carboxy-terminal cytoplasmic tail, respectively [1]. Unlike classical HLA molecules, MICA does not associate with -2-microglobulin and peptides derived from antigen processing but acts as a ligand for the activating NKG2D receptor mainly expressed by ␥␦ T cells, CD8⫹ ␣T cells, and natural killer (NK) cells, facilitating the recognition and elimination of virus-infected or transformed cells by the host immune system [2]. In contrast to other nonclassical HLA genes, MICA gene exhibits remarkable allelic diversity [3], MICA polymorphisms have been shown to be associated with a number of pathophysiological disorders, including malignancy, pathogen infection, autoimmune disease, and allograft rejection or graft-versus-host disease after allogeneic transplantation [4 –9].
* Corresponding author. E-mail address: [email protected] (W. Tian).
The Han Chinese represents about 94% of the population in China and could be categorized into Northern and Southern subgroups, which are separated approximately by the Yangtze River [10]. Hunan province is situated in the south of the middle Yangtze river valley and has a population of about 64 million (2000 census), in which Han ethnic group constitutes the largest ethnic group and comprises about 90% (⬇58 million) of the population in this region. There are also minority groups in this region, including Tujia, Miao, Dong, Yao, Hui, and others. According to the historical records, Hunan was inhabited by the ancestors of the modern Miao, Tujia, Dong, and Yao peoples until around 350 BC, when migration of Han Chinese from the central and north China occurred. Such migration continued for hundreds of years thereafter because of war or drought in the north. Currently, information on MICA alleles and MICA-containing HLA haplotypes in Chinese Han populations residing in different geographic locations is still limited. In a recent study, MICA allelic variation was investigated in a northern Chinese Han population [11]. In the present study, we investigated MICA allelic distribution in a Chinese Han population in Hunan province, in which a novel MICA allele, MICA*060, was identified. MICA*060 has an adenine substitution at position 862 in exon 4, which has only been recently
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.03.006
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reported for MICA*058 (http://hla.alleles.org/data/txt/mica_nuc. txt). As an extension of our study, we sought to determine the distribution of this adenine replacement in more diverse populations by screening additional 1432 samples. Unexpectedly, another novel MICA allele, MICA*062, was observed.
2.2. Extraction of genomic DNA from peripheral blood
2. Subjects and methods
A comprehensive strategy was used for MICA typing, as recently described by us [11]. Briefly, polymerase chain reaction-sequencebased typing (PCR-SBT) was used for the analysis of MICA exons 2, 3, and 4, fluorescent polymerase chain reaction–fragment analysis was used to determine the (GCT)n microsatellite at exon 5 of MICA gene (abbreviated as MICA-STR) [4,11], and polymerase chain reaction–sequence-specific priming (PCR-SSP) was applied to detect MICA gene deletion (MICA*Del) [4,11]. MICA allele assignment was based on the integrated information of exons 2–5.
2.1. Subjects The sample panel used for MICA population genetics consisted of 201 healthy, unrelated individuals of Han ethnicity. Among them, the subject carrying MICA*060 was a 44-year-old woman (sample ID: W124) (Fig. 1) living in northern China, whose maiden family had resided for the last three generations in Hunan province; the other 200 individuals were native residents in Hunan province. Among the 201 samples were 46 parental samples derived from 23 nuclear families. This sample panel comprised 59.2% men (119/ 201) and 40.8% women (82/201). The second sample panel comprised 1432 healthy, unrelated individuals, which were used to screen for the adenine substitution at position 862 in exon 4 (http://hla.alleles.org/data/txt/mica_nuc. txt). Among them were 1299 subjects of Han ethnicity in Hunan province, 67 subjects of ethnic minority (Hui, Miao, Tujia, Dong and Yao) in Hunan province, and 66 subjects of Han ethnicity in central and southeastern China. This sample panel consisted of 59.8% men (857/1432) and 40.2% women (575/1432). Ethnicity and ancestry was reported by each participant.A blood sample was taken with each person’s informed consent. There were no missing genotypic data. All protocols were approved by the Institutional Review Committee of local authorities.
Genomic DNA was extracted from 2 to 5 ml peripheral blood using a standard salting-out protocol [4]. 2.3. MICA typing
2.4. Screening of adenine substitution at position 862 in exon 4 A pair of sequence-specific PCR primers were designed to detect the adenine substitution at position 862 in exon 4 (http://hla.alleles. org/data/txt/mica_nuc.txt). The sequences of sense primer and antisense primer are 5=-ACATGGAACACAGCA-3= and 5=-ATGACTCTGAAGCAC-3=, respectively. The sense primer corresponds to the genomic segment from nucleotide position 8588 – 8602, and the antisense primer is complementary to genomic segment from nucleotide position 8743– 8757, when aligned with MICA consensus sequence available under accession number X92841. This pair of primers generate a 170-bp product when adenine substitution at position 862 in exon 4 is present. A pair of PCR primers were used as internal positive control, the primer sequences are 5=-TGCCAAGTGGAGCACCCAA-3= and 5=-GCATCTTGCTCTGTGCAGAT-3=, respec-
Fig. 1. Family segregation analysis of MICA*060. Haplotype is expressed in order of HLA-A-C-B-MICA-DRB1(from top to bottom). Samples W119 and W120 were typed only for HLA-A, C, B, and MICA loci; samples S103 and S104 were typed only for MICA locus. Samples W118, W119, and W120 were northern Chinese Hans: samples S102, S103, S104, and S105 were southern Chinese Hans residing in Hunan province; the proband, W124, currently resides in northern China.
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tively. This pair of primers amplify a 796-bp fragment from the third intron of HLA-DRB1 [12]. PCR amplification was carried out in a 10-l volume, containing 1X PCR buffer, 1.5 mmol/l MgCl2, 0.2 mmol/l dNTPs, 2.5 pmol of each SSP primer, 0.3 pmol of each internal control primer, 0.8 U of Taq polymerase (Promega, Shanghai, China), and 50⬃100 ng genomic DNA. A blank control, for which double-distilled water was used as DNA template, was included in each batch of PCR reactions. Amplification was performed using a MyCycler (Bio-Rad, Hercules, CA), cycling temperature profiles were as follows: the reaction mixture was subjected to one cycle of denaturation at 95⬚C for 2 minutes followed immediately by 30 cycles of 95⬚C for 20 seconds, 61⬚C for 60 seconds, and 72⬚C for 60 seconds, and a final extension at 72⬚C for 5 minutes before cooling to 15⬚C. PCR amplicons were identified by running 5 l of PCR product on a 2% agarose gel. The image of each gel was obtained using a bioimaging system (UVP, Upland, CA), and the size of the amplicons was determined by comparison against the migration of a 100-bp DNA ladder. 2.5. Cloning of exon 4 of 2 novel MICA alleles During MICA-sequence-based typing (SBT) of the 201 samples, sample W124 showed a novel sequence in exon 4, which differed from MICA*012:01 by an adenine substitution at position 862 or, alternatively, differed from MICA*002:01 by an adenine substitution at this position. To verify this polymorphism, PCR product of MICA exon 4 derived from sample W124 was therefore cloned using a TA cloning kit (TaKaRa, Dalian, China) [11]. The inserts of 20 randomly selected recombinant plasmids were sequenced in both directions. Two of the 1432 subjects, both of which were of Han ethnicity in Hunan province, were found to carry the adenine substitution at position 862 in exon 4. Interestingly, PCR-SBT indicated the existence of another new MICA allele, which was related to MICA*010. PCR products of MICA exon 4 derived from both samples (sample IDs: T156 and T450, respectively) were therefore cloned, and the inserts of 10 randomly selected recombinant plasmids from each sample were sequenced in both directions. 2.6. HLA typing HLA-A, C, and B data by generic PCR-SSP have been reported for the majority of the 201 samples [13,14]. In the present study, HLA-DRB1 together with HLA-A, C, and B loci of the remaining samples were typed by PCR-SSP using commercial typing reagents (Genovision, Westchester, PA). Subtyping of HLA-B*13, selected subtyping of HLA-B*48 and HLA-B*55 associated with the novel MICA alleles was accomplished by PCR-SSP using commercial subtyping kits (Invitrogen, Brown Deer, WI; Genovision, Westchester, PA). 2.7. Statistical analysis The program ARLEQUIN 3.11 (http://cmpg.unibe.ch/software/ arlequin3/) [15] was used to analyze the data, including the Hardy– Weinberg equilibrium (HWE) test, multilocus haplotype inference, and pairwise global linkage disequilibrium (LD). Maximum likelihood haplotype frequencies were estimated using the expectationmaximization (EM) algorithm, as implemented in the program ARLEQUIN 3.11. The classic coefficients of LD, ⌬, and of normalized LD, ⌬rel, were computed for each individual haplotype as described previously [11,14]. Fisher exact test was performed to determine the significance of ⌬rel. The LD analysis was restricted to HLA-BMICA haplotypes with observed frequencies ⬎1.5%. Statistical significance was defined at the 5% level after the p value was adjusted using Bonferroni’s correction, that is, by multiplying the p value by the number of independent comparisons performed.
3. Results 3.1. Hardy–Weinberg equilibrium test of MICA locus The MICA genotypic distributions were consistent with Hardy– Weinberg proportions (p ⫽ 0.1602). 3.2. MICA allele frequencies The MICA allele frequencies are summarized in Table 1. Nineteen MICA alleles were detected in this population, among which MICA*008:01 predominated with gene frequency of 30.35%. MICA*054, recently reported in a Caucasian individual [16], was observed in this population and associated with (GCT) 5 in exon 5. MICA*059, recently identified in a northern Chinese Han population [11], was also detected in this population. 3.3. LD between HLA-B and MICA All 13 kinds of HLA-B-MICA haplotypes with a frequency ⬎1.5% were in significant LD, among which 5 haplotypes showed a normalized LD value of 1 (Table 2). HLA-B*13:01 and HLA-B*13:02 displayed completely different LD patterns with MICA gene; conversely, multiple HLA-B lineages were also found in tight LD with the same MICA allele, for example, HLA-B*35, HLA-B*38 and HLAB*58 were all in strong LD with MICA*002:01. 3.4. HLA-A-C-B-MICA haplotypic diversity There were 23 kinds of HLA-A-C-B-MICA haplotypes occurring at least four times (with a frequency of ⱖ1%) (Table 3). Haplotypes HLA-A*02-C*01-B*46-MICA*010, HLA-A*11-C*07-B60-MICA*008: 01, and HLA-A*02-C*07-B60-MICA*008:01 were most prevalent, with frequencies of 14.5%, 9.16%, and 4.09%, respectively. Some haplotypes appeared to have derived from a common ancestral haplotypic block; for example, there were at least five kinds of HLA-B60-MICA*008:01 haplotypes with different HLA-A or HLA-C configurations. 3.5. HLA-A-C–B-MICA-DRB1 haplotypic diversity There were 26 kinds of HLA-A-C-B-MICA-DRB1 haplotypes occurring at least four times (with a frequency of ⱖ1%) (Table 4). HLA-A*02-C*01-B*46-MICA*010-DRB1*09, HLA-A*11-C*07-B60-
Table 1 MICA allele frequencies in 201 southern Chinese Han subjects MICA allelea
Frequency
*002:01 *004 *008:01 *008:02 *009:01 *009:02 *010 *011 *012:01 *017 *019 *020 *027 *033 *045 *054 *059 *Del *060
0.0895 0.0099 0.3035 0.005 0.0547 0.005 0.2462 0.0025 0.0796 0.005 0.087 0.0025 0.0274 0.0025 0.0672 0.0025 0.005 0.0025 0.0025
a
Using the current sequence-based typing (SBT) protocol, MICA *008:04 could not be distinguished from MICA *008:01; these two alleles differ at nucleotide position 21 in exon 1; MICA *049 could not be distinguished from MICA *009:01, these two alleles differ at nucleotide position 1067 in exon 6; MICA *048 could not be distinguished from MICA *027, these two alleles differ at nucleotide position 1017 in exon 5 (http://hla.alleles.org/data/txt/mica_nuc.txt).
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Table 4 Twenty-six kinds of HLA-A-C-B-MICA-DRB1 haplotypes
Table 2 Two-locus linkage disequilibrium analysis between HLA-B and MICA Haplotype
HF
⌬
⌬rel
pa
Haplotypea,b
Number
Frequency
B60-MICA*008:01 B*46-MICA*010 B*13:01-MICA*045 B75-MICA*019 B62-MICA*010 B*51-MICA*009:01 B*55-MICA*012:01 B*58-MICA*002:01 B*54-MICA*012:01 B*13:02-MICA*008:01 B*35-MICA*002:01 B61-MICA*027 B*38-MICA*002:01
0.2537 0.1815 0.0672 0.0521 0.0446 0.0398 0.0348 0.0348 0.0299 0.0274 0.0249 0.0224 0.0199
0.1759 0.1356 0.0622 0.0465 0.0299 0.0375 0.0314 0.0315 0.0273 0.0191 0.0227 0.0218 0.0181
0.9859 0.964 1 0.787 0.6644 0.938 0.8076 0.926 0.9181 1 1 1 1
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0037 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.0018 ⬍0.0001 ⬍0.0001 ⬍0.0001
*02-*01-*46-*010-*09 *11-*07-60-*008:01-*12 *02-*01-*46-*010-*08 *33-*03-*58-*002:01-*03 *11-*07-60-*008:01-*08 *11-*08-75-*019-*12 *30-*06-*13:02-*008:01-*07 *02-*07-60-*008:01-*08 *11-*07-60-*008:01-*15 *11-*04-62-*010-*04 *02-*07-60-*008:01-*04 *02-*03-*13:01-*045-*12 *24-*01-*54-*012:01-*04 *11-*07-60-*008:01-*11 *11-*03-60-*008:01-*09 *24-*08-75-*019-*12 *02-*07-60-*008:01-*14 *11-*08-75-*019-*15 *02-*01-*46-*010-*11 *02-*07-*38-*002:01-*08 *11-*01-*55-*012:01-*04 *11-*01-*55-*012:01-*09 *11-*03-*13:01-*045-*12 *24-*03-*13:01-*045-*15 *24-*07-60-*008:01-*09 *11-*03-60-*008:01-*14
35 15 13 9 8 8 8 6 6 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4
0.0875 0.0371 0.0334 0.0224 0.0206 0.0202 0.0199 0.0155 0.0136 0.0133 0.0124 0.0124 0.0119 0.0115 0.0112 0.0111 0.011 0.0103 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.0098
All 13 HLA-B-MICA haplotypes (with a frequency ⬎1.5%) showed values of p ⬍0.00385 (0.05 after Bonferroni’s correction) and were considered to be in significant linkage disequilibrium.
a
MICA*008:01-DRB1*12 and HLA-A*02-C*01-B*46-MICA*010DRB1*08 were the most common haplotypes with frequencies of 8.75%, 3.71%, and 3.34%, respectively. Diversification at HLA-DRB1 locus was found in several common HLA-A-C-B-MICA haplotypes, for example, HLA-A*02-C*01-B*46-MICA*010 was linked to at least three different HLA-DR serologic groups. By contrast, some haplotypes appeared to be conservatively maintained, for instance, haplotype HLA-A*30-C*06-B*13:02-MICA*008:01 was exclusively linked to HLA-DRB1*07.
a
Haplotype is expressed in order of HLA-A-C-B-MICA-DRB1. Only haplotypes occurring at least 4 times (with a frequency of ⱖ1%) are presented.
b
3.6. MICA*060 Among the 20 randomly selected recombinant plasmids, six clones showed a novel sequence (tentatively named MICA*TWFHnew), which when aligned with MICA*012:01, has an adenine substitution at position 862 in exon 4, leading to an amino acid change at codon 265 from Glycine (GGG, MICA*012:01) to Arginine (AGG, MICA*TW-FHnew); six clones had a sequence identical to MICA*012:01; five clones had sequences each differing from MICA*TW-FHnew by one to three mutations at positions other than 862 in exon 4; 3 clones had sequences each differing from MI-
Table 3 Twenty-three kinds of HLA-A-C-B-MICA haplotypes
CA*012:01 by one to two mutations at positions other than 862 in exon 4. Information collected from sequences in exons 2 and 3 suggested a MICA*002/MICA*012 heterozygote. Because MICA* 002:01 and MICA*012:01 share sequence in exon 4, which means that MICA*TW-FHnew could be derived from either of them, family analysis was carried out to resolve the ambiguity. As shown in Fig. 1, MICA*TW-FHnew segregated with sequence motifs of MICA*012:01 on an HLA-A*02-C*01-B*55:02 -DRB1*14 haplotype. The name MICA*060 has been officially assigned to MICA*TWFHnew by the WHO Nomenclature Committee in February 2010. The GenBank accession number for MICA*TW-FHnew is FJ556895. MICA*060 was associated with (GCT) 4 in exon 5.
Haplotypea,b
Number
Frequency
3.7. MICA*062
*02-*01-*46-*010 *11-*07-60-*008:01 *02-*07-60-*008:01 *11-*03-60-*008:01 *11-*08-75-*019 *24-*07-60-*008:01 *11-*03-*13:01-*045 *33-*03-*58-*002:01 *24-*03-60-*008:01 *11-*01-*55-*012:01 *30-*06-*13:02-*008:01 *11-*01-*46-*010 *02-*07-*38-*002:01 *02-*03-*13:01-*045 *11-*01-*54-*012:01 *11-*04-62-*010 *24-*14-*51-*009:01 *24-*01-*46-*010 *02-*15-*51-*009:01 *24-*08-75-*019 *24-*01-*54-*012:01 *24-*03-61-*027 *24-*03-*13:01-*045
58 37 16 14 14 13 13 12 8 8 8 7 7 6 5 5 5 4 4 4 4 4 4
0.145 0.0916 0.0409 0.0352 0.0343 0.0325 0.0323 0.0299 0.0209 0.0199 0.0199 0.018 0.0174 0.0153 0.0136 0.0127 0.0121 0.0111 0.01 0.0098 0.0096 0.0095 0.0094
Analyses of the 10 randomly selected recombinant plasmids derived from PCR product of MICA exon 4 of each sample indicated that this new MICA allele (tentatively named MICA*NEW-SY) had sequence identical to MICA*058 in exon 4. When aligned with MICA*010, MICA*NEW-SY has an adenine substitution at position 862 in exon 4, leading to an amino acid change at codon 265 from glycine (GGG, MICA*010) to arginine (AGG, MICA*NEW-SY). The name MICA*062 has been officially assigned to this new allele by the World Health Organization (WHO) Nomenclature Committee in June 2010. The GenBank accession number for MICA*NEW-SY is HM019306. The genotype of sample T156 was as follows: HLAA*02, A*11; C*01, C*08; B*46, B*48:01; MICA*010, MICA*062; MICA*A5, -; DRB1*11, DRB1*14. The genotype of sample T450 was as follows: HLA-A*02, A*33; C*03, C*08; B*48:01, B*58; MICA*002: 01, MICA*062; MICA*A5, MICA*A9; DRB1*03, DRB1*14. MICA*062 was associated with (GCT) 5 in exon 5. The naming of MICA*060 and MICA*062 follows the agreed-on policy that, subject to the conditions stated in the most recent Nomenclature report [17], names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature report.
a
Haplotype is expressed in order of HLA-A-C-B-MICA. Only haplotypes occurring at least four times (with a frequency of ⱖ1%) are presented. b
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W. Tian et al. / Human Immunology 72 (2011) 510-515
4. Discussion This study was designed to investigate MICA genetic diversity in a southern Chinese Han population by sequence-based typing, for which no such data had previously been available. Our data revealed a high degree of MICA allelic diversification in this population. Similar to most other ethnic groups studied [11,18-21], MICA*008:01 was the most prevalent allele in this population, in contrast to two tribal groups in sub-Saharan Africa [22] and the American Indian populations [23,24], where MICA*002:01, MICA* 020 or MICA*027 predominated. The two most common alleles, MICA*008:01 and MICA*010, combined accounted for about 55% of the MICA gene pool. A large number of MICA alleles were observed only sporadically in this population. HLA-B*13 lineage displayed a clear-cut dichotomy of association with MICA alleles in this southern Chinese Han population, confirming our earlier finding in a northern Chinese Han population [11]. Two individuals in this southern Chinese Han population were found to carry HLA-B*13:01-MICA*059 haplotype. These findings may help to substantiate the notion of southward Chinese Han migration in history. The consistent dichotomous association between HLA-B*13 lineage and MICA alleles is of particular importance to allogeneic transplantation in Chinese Han populations, as evidence is accumulating about the role of MICA in transplant rejection [25]. Subjects with HLA-B*13 may require MICA allelic typing to optimize selection of allogeneic donors. Ten kinds of HLA-B-MICA haplotypes were in significant LD in both the northern and southern Chinese Han populations. However, the distribution of some haplotypes showed a high degree of variability between these 2 groups. For instance, the frequency of haplotype HLA-B60-MICA*008:01 was about sixfold higher in the south (0.2537) than in the north (0.0385) [11]; conversely, the frequency of haplotype HLA-B*13:02-MICA*008:01 decreased southward (0.0962 in the north vs 0.0274 in the south). MICA*012:01, which appeared to be very infrequent in the northern Chinese Han population [11], showed a noticeable frequency in the southern Chinese Han population. Significant LDs of MICA*012:01 with HLA-B*54 and HLA-B*55 were detected in the southern Chinese Han population. Relatively high frequency of MICA*012 has only been reported in very few populations, including Japanese [19], Korean [26], and a population of Jewish descent [21]. LD between MICA*012 and HLA-B*54 or HLA-B*55 was also found in those populations, indicating a common ancestral origin. The fact that MICA*012:01 was found on both HLA-B*54 and B*55 may also reflect mutational origin of the two HLA-B alleles on a single MICA haplotype. A new MICA allele, MICA*060, was observed on an HLA-A*02-Cw*01-B*55:02 -DRB1*14 haplotype in the southern Chinese Han population, suggesting that MICA*060 has probably arisen from MICA*012:01 through a single point mutation. MICA locus displays a G/A dimorphism at position 862 in exon 4 (http://hla.alleles.org/data/txt/mica_nuc.txt). Among the 71 MICA alleles, only MICA*058 and MICA*060 have an adenine at this position. MICA*058, recently described in a Hispanic individual (http:// hla.alleles.org/data/refs/mica_refs.html), was not observed in the southern Chinese Han population. Instead we uncovered another novel MICA allele, MICA*062, which has possibly derived from MICA*010 through a single mutation event. Of particular interest is that MICA*062 was harbored on an HLA-C*08-B*48:01-DRB1*14 haplotypic segment in this southern Chinese Han population, because HLA-B*48 has been consistently shown to be primarily linked to MICA gene deletion (MICA*Del) in east Asian populations [11,19,27]. MICA*054, differing from MICA*010 by a guanine at nucleotide position 871 in exon 4, was also detected in this southern Chinese Han population. All these findings may indicate a relatively rapid evolution of MICA*010 in exon 4.
The inclusion of HLA-C locus helped refine HLA haplotype(s) specific to Chinese sub-populations. For example, HLA-A*11-B60MICA*008:01 was much more common in the southern Chinese Han population (0.1268, Table 3) than in the northern Chinese Han population (0.0192) [11]. A close look revealed that while HLAA*11-C*03-B60-MICA*008:01 showed comparable frequency in these 2 populations (0.0352 vs 0.0192), HLA-A*11-C*07-B60MICA*008:01 was virtually undetectable in the northern Chinese Han population [11], suggesting that this haplotype is highly characteristic of southern Chinese Han population. To our knowledge, this is the first study of its kind to construct HLA-A, C,B, MICA and DRB1 haplotypic repertoire in a single investigation. The multi-locus haplotype data here may provide a unique resource for future studies between human populations. Our study has specific clinical implication in HLA-linked disease association in populations of Chinese ancestry. HLA gene complex has been consistently shown to be associated with nasopharyngeal carcinoma (NPC) [28,29], an epithelial malignancy with the highest incidence rate in southern Chinese populations. Particular HLA-B specificities, mainly including HLA-B*38, B*46 and HLA-B*58 confer high risk for NPC in Chinese populations [30,31]. Evidences are emerging suggesting the involvement of MICA-STR and MICA-129 polymorphism in the susceptibility to NPC [4,32]. Thus, this investigation establishes the extent of linkage disequilibrium between MICA alleles and HLA-B locus, findings shown here are valuable for future haplotype mapping aimed at refining the primary susceptibility locus to NPC, which is most likely located in HLA-B subregion in southern Chinese populations. In conclusion, our data demonstrated a high degree of MICA gene diversity in a southern Chinese Han population. Our study unraveled the peculiarity in terms of MICA allelic and haplotypic repertoire and identified two novel MICA alleles in this population. Findings shown here are of importance for future studies of the potential role of MICA in allogeneic organ transplantation and HLAlinked disease association in populations of Chinese ancestry, and will inform studies of evolution of the MHC gene complex. Acknowledgments We thank all of the blood donors. We also express our gratitude to LiXin Li, Fan Wang, and JunJie Wang for their assistance in the early stage of this project. This work was supported by grants from the Program for New Century Excellent Talents in University (Proj.No. NCET-08-0564), National Natural Science Foundation of China (Proj.No.30671915, 30300311), The Outstanding Youth Science Foundation of Hunan Province (Proj.No.04JJ1007) and Central South University (Proj.No. ZKJ2010039). References [1] Bahram S, Bresnahan M, Geraghty DE, Spies T. A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 1994;91:6259 – 63. [2] Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 1999;285: 727–9. [3] Robinson J, Malik A, Parham P, Bodmer JG, Marsh SG. IMGT/HLA database ⫺ a sequence database for the human major histocompatibility complex. Tissue Antigens 2000;55:280 –7. [4] Tian W, Zeng XM, Li LX, Jin HK, Luo QZ, Wang F, et al. Gender-specific associations between MICA-STR and nasopharyngeal carcinoma in a southern Chinese Han population. Immunogenetics 2006;58:113–21. [5] Kirsten H, Petit-Teixeira E, Scholz M, Hasenclever D, Hantmann H, Heider D, et al. Association of MICA with rheumatoid arthritis independent of known HLADRB1 risk alleles in a family-based and a case control study. Arthritis Res Ther 2009;11:R60. [6] Boukouaci W, Busson M, Peffault de Latour R, Rocha V, Suberbielle C, Bengoufa D, et al. MICA-129 genotype, soluble MICA, and anti-MICA antibodies as biomarkers of chronic graft-versus-host disease. Blood 2009;114:5216 –24. [7] Shojima J, Tanaka G, Keicho N, Tamiya G, Ando S, Oka A, et al. Identification of MICA as a susceptibility gene for pulmonary Mycobacterium avium complex infection. J Infect Dis 2009;199:1707–15.
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