| Background MICA (major histocompatibility complex class I chain-related gene A) is non-classical HLA(human leukocyte antigen)molecular and its gene locates at the human chromosome 6p21.3.Studies was approved that MICA is similar to MHC class I gene, butexists obvious differences. The ultimate difference lies in thatMICA neither binds withβ2 microglobin protein nor presents antigenpeptide, but it can bind with NKG2D/DAP10 receptor to regulate thefunction of NK(natural killer) and T cell. MICA has highpolymorphism as classical HLA class I, it has reported 64 allelesso far. The polymorphism of MCIA mainly is in exons 2 to 5, partlyin exon 1 and 6. Exon 5 of the MICA gene contains a polymorphismshort tandem repetition sequence(GCT), in present, eight alleleshave been reported as A4,A5,A6,A7,A8,A9,A10 and A5.1, of whichthe number with A presents the repeat times of GCT fragment. A5. 1presents a G insertion after the second GCT of A5. Studies reportedMICA is related with some autoimmune diseases and tumor. It wasapproved recently when the anti-HLA and anti-MICA antibodies ofpatients are negative at the same time, the patient survival rateof solid organ transplantation is more higher, but the functionof MICA is still unclear. We will establish precise MICA genotyping method,and acquire the MICA polymorphism data in the Zhejiang Hanpopulation, which will facilitate to further study the functionof MICA. Now the mainly MICA genotyping method is the PCR sequencingfor exon 2 to 4 directly with typing the polymorphism of exon 5by GeneScan technique, or typing the polymorphism of exon 5 byGeneScan technique alone in home and commercial reagent, which cannot discriminate all MICA allele and lead to some ambiguous results.In our study,we will sequence exon 2 to 6 directly and also typingexon 5 by Genescan technique. We will establish a systemic,comprehensive,accurate genotyping method for MICA, which it isfirstly to analyze the sequences of MICA exon 5 and 6 in thepopulation. Our method will be perfected previous MICA genotypingmethod and acquire polymorphism data of MICA in zhejiang Hanpopulation, which can provide scientific basis for studying thefunction of MICA in the future.Methods1,Samples:Peripheral blood were collected from 100 unrelatedhealthy donors after their consent. We collected 5 ml blood withEDTA anticoagulant and reserved them below -20℃for study.2,DNA extraction: Genomic DNA was isolated from EDTA blood samplesby Pel-Freeze DNA commercial extraction kit according tomanufacturer's protocol.3,Exon 2-6 of MICA sequence analysis3.1 PCR amplification: the primers sequences are refered to MICAgene sequences, two pairs of primers were used to amplify for exon2-6. The first pair of primers is amplified for exon 2-5,the otherpair of primers is amplified for exon 5-6. The primers sequencesand amplification conditions refer to the text as below, the volumeof PCR amplification is 25ul.3.2 PCR product purification: To degrade excess primers andnucleotides from the extension PCR product for the subsequent sequencing reaction,5 units exonuclease I (Exo I) and 2 unitsshrimp alkaline phosphatase (SAP) were applied to each 25μl of PCRproduct. The mixture was at 37℃for 30 minutes, followed by 15minutes at 80℃in ABI 9700 PCR instrument.3.3 sequencing reaction: sequencing reactions for both directionsfrom exon 2 to 6 were carried out using an ABI BigDyeTM TerminatorCycle Sequencing Kit according to manufacturer's protocol. Thesequencing reaction conditions include an initial denaturationstep at 95℃for 5 minutes, followed by 30 cycles at 95℃for 30seconds, 50℃for 10 seconds,60℃for 4 minutes, and cool to 4℃in ABI 9700 PCR instrument.3.4 sequencing reaction product purification and electrophoresisanalysis: we applied ethanol/acetate sodium method to purify theproducts with electrophoresis analysis in ABI 3730 sequencer.3.5 Analysize and assign the result: Assign 3.5 software and MTNavigator software were adopated for sequence result analysis.Thefinal result of MICA was assigned by Assign 3.5 software.4,Microsatellite polymorphism in exon 5 of MICA by GeneScan4.1 GeneScan PCR amplification: The primers sequences refer todocument.The sense primer was labelled with the fluorescent dyeFAM at the 5' end. The primers sequences and amplificationconditions refer to the text, the volume of PCR amplification is20μl.4.2 GeneScan test: The amplication product was diluted with 9 timeswater,then 5μl diluted product was mixed with 4.8μl formamide and0.2μl internal standard ROX500. The mixture was denatured at 95℃for 4 minutes, then electrophoresis analysize in ABI 3730sequencer with GeneScan module.4.3 GeneScan result analysis: GenMapper v3.7 software was used foranalysize the result.We calucate the GCT number according to thefragment length, and validate the GeneScan results with sequencingmethod.5,MICA*Del detection: We detected MICA*Del allele by PCR-SSP, the primers refer to document, one pair of primers amplify the MICA*Delconsensus sequence,the other pair amplify conserved homologoussequences in the human growth hormone (HGH) gene as internal control.The primers sequences and amplification conditions refer to thetext as below, the volume of PCR amplification is 10μl.6,HLA-B was detected by PCR-SBT,which was established in ourlaboratory previously.7,Statistics analysis: Gene frequences(GF) were caculated bydirect counting,the formula is allele numbers/2N(N=100). weanalysized the haplotype frequency(HF) and linkage disequilibration (LD) by ARLEQUIN 3.01 software.Hardy-Weinberg equilibriumfollowed the procedure described in Guo and Thompson,p<0.05 issignificance.Results1,PCR sequence-based typing: The exon 2 to 6 sequences of MICA canbe amplified by two pairs of primers, which can obtained specificPCR amplification fragments respectively. Clearly sequence figurecan be acquired with bidirectional sequencing reaction and the MICAalleles of samples can be assigned by software automately. ThePCR-SBT for MICA which we establish is feasible.2,Standard sequence analysis: We confirmed the sequences of MICA*019and have submitted the sequences to Genbank, which the accessionnumber is EU267 602. We corrected the mistake MICA*019 (1073bp T→A)in WHO nominated database and received the confir -mation letterfrom WHO nominate committee.3,The MICA allele distribution in Zhejiang Han population: Weobtained 14 MICA alleles in zhejiang Han population, the mostfrequent allele was MICA*00801/04. The allele frequencies were MICA*00801/04 (27.0%), MICA*010 (18.5%),MICA*00201 (15.5%),MICA*019 (9. 5%),MICA*00901 (8.0%),MICA*027 (7.0%),MICA*01201 (5.0%),MICA* 049 (3.5%),MICA*045 (1.5%),MICA*00802 (1.5%),MICA*00701 (1.0%),MICA*Del (1.0%),MICA*004 (0.5%),MICA*00902(0.5%) respectively. By statistical analysis, observed heterozygosis was 0. 83, expected heterozygosiswas 0.84,p=0.75.4,The STR of MICA exon 5 distribution in Zhejiang Han population:We found 5 different GCT numbers: A4,A5,A5.1,A6,A9, the most frequentallele was A5. The allele frequencies were A5 (35%),A5.1 (28.5%),A4(7.5%),A6 (14.5%),A9 (13.5%),Del (1.0%) respectively. Bystatistical analysis, observed heterozygosis was 0.74, expectedheterozygosis was 0.749,p=0.96.5,HLA-B gene distribution in Zhejiang Han population:we detected40 alleles in total, which were B*4001 (11.5%),B*4601 (9%),B*5101(6.5%),B*5801 (6.5%),B*1502 (6%),B*1501 (5%),B*1302 (4.5%),B*5201(3.5%),B*0702(3%),B*1511 (3%),B*4006 (3%),B*5407(3%),B*3501(2.5%),B*3802 (2.5%),B*4002 (2.5%),B*5502 (2.5%),B*3701 (2%),B*3901 (2%),B*4402(2%),B*0705(1.5%),B*0801 (1.5%),B*1301 (1.5%),B*1527(1.5%),B*2704(1.5%),B*3503(1.5%),B*4403 (1.5%),B*5102 (1.5%),B*2705 (1%),B*4801 (1%),B*1507(0.5%),B*1505(0.5%),B*1513 (0.5%),B*2706(0.5%),B*2707(0.5%),B*3505(0.5%),B*3801(0.5%),B*3905(0.5%),B*5001(0.5%),B*6701(0.5%),B*8101(0.5%) respective -ly. By statistical analysis,observed heterozygosis was 0.97, expected heterozygosis was0.95,p=0.33.6,The haplotype frequency of MICA and HLA-B gene and the linkagedisequilibrium analysis: By Arlequin 3.01 software, there were 58haplotypes in the population, which were MICA*00801-B*4001 (11.5%),MICA*010-B*4601 (8.0%),MICA*00201-B*5801 (6.5%),MICA*019-B*1502 (5.0%),MICA*00801-B*1302 (4.0%),MICA*010-B*1501 (4.0%),MICA*049 -B*5101 (3.5%),MICA*01201-B*5401 (3.0%),MICA*00901-B*5201 (3.0%),MICA*010-B*1511 (3.0%),MICA*027-B*4006(3.0%). The frequencies of MICA*00201-B*3501,MICA*00201-B*3802,MICA*00801-B*0702,MICA*00901-B*5101 were 2. 5% respectively. Thefrequencies of MICA*01201 -B*5502,MICA*00801-B*3701,MICA*00801-B*4402were 2.0% respective -ly. The frequencies of MICA*00201-B*3503,MICA*00801-B*0801,MICA* 00801-B*3901,MICA*00802- B*0705,MICA*010-B*1527, MICA*019- B*2704,MICA*027-B*4002 were 1.5% respectively.Thefrequencies of MICA*Del-B*4801,MICA*019-B*1501,MICA*00901-B*4403,MICA*00901 -B*5102,MICA*045-B*1301 were 1.0% respectively.Thefrequencies of MICA*00201- B*3505,MICA*00201-B*3801,MICA*00201-B*3901,MICA* 00201- B*5201,MICA*00201-B*6701,MICA*004-B*4403,MICA*00701-B*1301,MICA*00701 -B*2705,MICA*00801-B*2706,MICA*00801 -B*5502,MICA*00801-B*3905,MICA*00801-B*4002,MICA*00901-B*4001,MICA* 00902-B*5001,MICA*010 -B*1505,MICA*010-B*2705,MICA*010-B*2707,MICA*010-B*8101,MICA*019-B*4001,MICA*019-B*4002,MICA*019-B*4601,MICA*019-B*5102,MICA*027-B*1502,MICA*027-B*1513,MICA*027-B*1527,MICA*027-B*4601,MICA*027-B*5101,MICA*045-B*1302were 0.5%respectively. Linkage disequilibrium analysis showed that theywere associated strongly, with MICA*00801-B*4001 (D=0.081, D′=0.8 90,r~2=0.31, X~2=61.24,p<0.00l),MICA*010-B*4601 (D=0.063, D′=0.863,r~2=0.32, X~2=65.00,p<0.00l),MICA*00201-B*5801 (D=0.055, D′=1.000,r~2=0.38, X~2=75.80,p<0.00l),MICA*019-B*1502 (D=0.045, D′=0.899,r~2=0.45, X~2=89.73,p<0.00l),MICA*00801-B*1302 (D=0.028, D′=0.848,r~2=0.092, X~2=18.31,p<0.001),MICA*010-B*1501 (D=0.031, D′=0.755,r~2=0.13, X~2=26.41,p<0.001) . The linkage analysis data of thehaplotype below 4.0% refer to the text.7,The relationship of the repetition numbers in exon 5 and HLA-Bgene:By Arlequin 3.01 software, there were 48 haplotypes in thepopulation, with A5.1-B*4001 (11. 5%),A5-B*4601 (9%),A9-B*5801 (6.5%),A5-B*1502(5.5%),A6-B*5101(5.5%),A5-B*1501 (5.0%),A5. 1-B*1302 (4%),A4-B*5401 (3%),A5-B*1511 (3%),A5-B*4006 (3%),A6-B*5201 (3%),A5.1-B*0702(2.5%),A9-B*3501 (2.5%),A9-B*3802 (2.5%),A4- B*5502 (2%),A5-B*1527(2%),A5-B*4002 (2.0%),A5.1-B*3701 (2%),A5. 1-B*4402 (2.0%),A4-B*1301(1.5%),A5-B*2704 (1.5%),A5. 1-B*0705 (1.5%),A5.1-B*0801 (1.5%),A5.1-B*3901 (1.5%),A6-B*4403 (1.5%),A6-B*5102 (1.5%),A9-B*3503 (1.5%),A5-B*5101(1%),Del-B*4801(1%),A4-B*1302(0.5%),A4-B*2705(0.5%),A5-B*1505(0.5%),A5-B*1513 (0.5%),A5-B*2707 (0.5%),A5-B*2705 (0.5%),A5-B*4001 (0.5%),A5-B*8101 (0.5%),A5. 1-B*2706 (0. 5%),A5.1-B*3905 (0.5%),A5.1-B*4002 (0.5%),A5.1-B*5502(0.5%),A6-B*4001 (0.5%),A6-B*5001 (0.5%),A9-B*3505 (0.5%),A9-B*3901 (0.5%),A9-B*3801 (0.5%),A9-B*5201 (0.5%),A9-B*6701 (0.5%) respectively. Linkage disequilibrium analysisshowed they were associated strongly,with A5. 1-B*4001 (D=0.078,D′=0.887,r~2=0.27, X~2=53.66, p<0.001),A5-B*4601 (D=0 . 059, D′=1.000,r~2=0.18,X~2=36.73,p<0.001),A9-B*5801 (D=0.055, D′=1.000, r~2=0.38,X~2=75.80,p<0.00l), A5-B*1502 (D=0.036,D′=1.000,r~2=0.11, X~2=21.62,p<0.001),A6-B*5101 (D=0.047,D′=0.824, r~2=0.33, X~2=66.11, p<0.00l),A5-B*1501 (D=0.033,D′=1.000,r~2=0.098, X~2=19.55, p<0.00l) . Thelinkage analysis data of the haplotype below 5.0% refer to thetext.8,MICA-Del allele detection:Two samples with HLA-B*4801 were MICA-Delpositive(allele frequency is 1%)and other samples were negativein 100 Zhejiang Han population. We also found six samples wereMICA*Del positive in 8 samples with HLA-B*4801 by chosen. We confirmedMICA-Del allele in Zhejiang Han population, which associate withHLA-B*4801.Conclusion1. The PCR-SBT for sequencing exon 2-6 and GeneScan technique forexon 5 that we established in this study is feasible.2. We confirmed the sequences of MICA*019, and received thecertificate letter from WHO nominate committee.3. We acquired the polymorphism data of MICA gene in Zhejiang Hanpopulation.4. We obtained the linkage heredity data between MICA and HLA-Bin the allele level.5. We acquired the linkage heredity data between MICASTR and HLA-Bin the allele level.6. We confirmed that MICA-Del allele exists in Zhejiang Han population, which associates with HLA-B*4801 allele. |
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