Original Papers Hum Hercd 1991;41:209-214
© 1991 S.K arger A G . Basel 0001-5652/91/0414-0209 S 2.75 II
Frequency of HLA-DQA1 Alleles in the Japanese Population Keiji Tamakia, Toshimichi Yamamotoa, Rieko Uchihi", Yoshinao Katsumata3, Keiko Kondob, ShinichiMizunob, AkinoriKimurac, Takehiko Sasazukic a Department of Legal Medicine, Nagoya University School of Medicine, Nagoya; h Japanese Red Cross Aichi Blood Center, Seto-shi; c Department of Genetics, Medical Institute of Bioregulation. Kyushu University. Fukuoka. Japan
Key Words. DNA polymorphism • HLA-DQA1 locus • Polymerase chain reaction Abstract. One of the HLA class II genes, HLA-DQAi, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQAI gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQAI ge notype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the num bers expected under the Hardy-Weinberg equilibrium. The HLA-DQAI genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.
(PCR) procedure [1] allows the enzymatic amplification of a specific segment of hu The HLA class II molecules DR. DQ man genomic DNA and direct detection of and DP are highly polymorphic cell sur the allelic variation in the HLA-DQA lo face molecules which have an important cus [2, 3], The DQA genotypes of single function in the generation and regulation hairs can be detected using sequence-spe of the immune response. The elucidation cific oligonucleotide (SSO) probes [4], It of their polymorphisms is thus important has also been suggested that the allelic for understanding the mechanism of allo- variation in the HLA-DQA locus detected rccognition and the generation of autoim by SSO probes was greater than the poly mune diseases. These polymorphisms are morphisms defined by serological typing usually detected serologically using vari [ 21. ous antibodies. However, recent develop Recently, the WHO Nomenclature for ment of the polymerase chain reaction Factors of the HLA System Committee
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 8/12/2018 11:11:17 PM
Introduction
210
Tamaki/Yamamoto/Uchihi/Katsumata/Kondo/Mizuno/Kimura/Sasazuki
No.
DQA1 specificity
Sequence (5-3')
1 2
0101 0102, 0103, 0501 0101. 0102. 0201, 0301 0103 0201 0301 0501 0401. 0601 0101, 0102, 0401, 0501 0103, 0201. 0601
CACGTAGAACTCCTCATC
3 4 5 6 7 8 9 10
CACGTAGAACTGCTCATC CTCCTTCCTCTCCAGGTC GTCTCCTTCTTCTCCAGG CAGAGGCAACTTCCAGAC ATCTTCTAAATCTGCGGA TCAGACTGTTCAAGTTAT CGTAGAACTGCTCGTCTC TCATGGGTGTACTGGCCA TCATGGGTGAACTGGCCA
proposed the nomenclature of HLA al leles and loci defined by nucleotide or in ferred amino acid sequences [5]. Under the nomenclature, the HLA-DQA1 locus has 8 defined alleles as DQA1*0101, DQA 1*0102, DQA1*0103. DQA1*0201, DQA1*0301, DQA 1*0401, DQA1*0501 and DQA1*0601. However, the gene frequen cies of each allele of HLA-DQA1 have not been reported so far. The following report describes the gene frequencies of HLADQA1 in the Japanese population using a set of SSO probes. Furthermore, HLADQA1 genotypes were determined with aged bloodstains using the present method. Materials and Methods Extraction o f DNA from Blood Blood samples were obtained from 290 healthy Japanese and mixed with heparin in small test tubes. They were centrifuged and the huffy coat was col
lected. The huffy coat was rinsed twice with 2 vol of a solution containing 0.01 M Tris-HCI (pH 7.6). 0.005 M MgCI, and 0.01 M NaCl. then resuspended in one third volume of a solution containing 0.01 M TrisHCI (pH 7.6). 0.01 M EDTA (pH 8.0). 0.05 M NaCl. 0.5% (wlx) sodium dodecyl sulfate (SDS) and 200 gg/ml proteinase K (Sigma Chemical. St. Louis, Mo.. USA). Actual solvent volumes were dependent on the volume of blood used. The final suspension was incubated overnight at 42 °C. DNA in the specimens was extracted twice with phenol/chloroform (1:1) and once with chloroform, and was then precipitat ed with isopropanol. The precipitation was rinsed 3 times with 70% (w/v) ethanol, dried under vacuum, dissolved in a solution containing 0.01 M Tris-HCI (pH 7.4) and 0.001 M EDTA (pH 8.0) and stored at 4°C until required for use. The concentration of DNA was determined by measuring the optical den sity at 260 nm. DNA was extracted from bloodstains according to the protocol described by Gill et al. [6], PCR Amplification o f DNA DNA (1 gg) was amplified by PCR using the fol lowing primers: GH26 (5'-GTGCTGCAGGTGTAAACTTGTACCAG-3') and GH27 (5'-CACGGATCCGGTAGCAGCGGTAGAGTTG-3') |7|. Two units of Taq DNA polymerase (Perkin-ElmerCetus, Norwalk. Conn., USA) and 20 pmol of each primer were added to polymerase buffer (100 gl) containing 10 mM Tris-HCI (pH 8.4), 50 mM KCI. 2.5 mM MgC!,, gelatin (1(H) gg/ml) and 200 gAJ each of the four dcoxyribonucleoside 5'-triphosphatcs (dATP. dCTP. dGTP and TTP). 40 cycles of PCR were carried out using an automated device (DNA Thermal Cycler. Perkin-Elmer-Cetus), and each cy cle was performed for 2 min at 94. 55 and 72 °C. Af ter the last cycle, the samples were incubated for an additional 5 min at 72 °C. The amplified DNA was checked using a gel of 3% (w/v) NuSieve (FMC, Rockland, Me., USA) stained with ethidium bro mide. Preparation o f Labeled SSO Probes Ten SSO probes were devised as described by Fukui et al. [8] (table I) based on published HLADQA1 alignments [3, 9-15], They were synthesized by a 381A DNA Synthesizer (Applied Biosystems, Foster City, Calif.. USA). Following a conventional purification procedure, probe concentrations were determined by measurement of absorbance at
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 8/12/2018 11:11:17 PM
Table 1. SSO probes used in the present study
211
Frequency of HI.A-DQAI Alleles in Japanese
DQA1
SSO °1 •
«
•
• •
2 3 •
V
•
•
•
•
4 •
•
•
•
•
4
©
•
•
r
•
•
• •
5 • 6 • 7
•
8
•
9
•
10
•
260 nm. 30 pmol of each oligonucleotide were la beled with digoxigenin-ll-dUTP (2.5 nmol; Boehringer-Mannheim. Mannheim, FRG) in a labeling buffer [140 \iM sodium cacodylate, 30 mM Tris-FICI (pH 7.2). I mM CoCI,. 100 gM dithiothreitol] and 20 units of terminal transferase for 2 h at 37 °C. Hybridization and Detection o f HLA-DQA 1 Genotypes All the following procedures were performed at room temperature unless otherwise stated. A nylon membrane (Hybond N+. Amersham. Amersham, UK) of suitable size was completely soaked in 10 x SSPF for 10 min: I x SSPE is 0.15 M NaCl, 0.01 M so dium phosphate and 1 mW EDTA (pH 7.4). Ampli fied DNA (5 pi) was applied to a membrane in a Bio-Dot microfiltration apparatus (Bio-Rad. Rich mond. Calif.. USA). After drying completely, the membrane was soaked in a solution containing 0.4 M NaOH for 1 min and then in 10 x SSPE for 5 min, and dried completely again. Membranes were prcincubated in 6x SSPE, 0.1% (w/v) sodium N-lauroyl sarcosinate, 0.02% (w/v) SDS and 5 x Denhardt’s solution: 1 x Dcnhardt's solution is 0.02% (w/v) Ficoll 400 (Pharma
•
•
1
•
• •
o
•
•
• •
•
•
•
•
•
•
•
•
•
•
•
•
•
cia, Uppsala, Sweden), 0.02% (w/v) bovine serum al bumin (Fraction V, Wako Pure Chemical, Osaka. Ja pan) and 0.02% (w/v) polyvinylpyrrolidone, for 20 min at 42°C. Hybridization was performed in the same solution as prehybridization containing 2 nM SSO probes for 20 min at 42°C. After hybridization the membranes were washed for 5 min with 2x SSPE, then soaked in a solution containing 3.0 M tétraméthylammonium chloride, 50 mM Tris-HCI (pH 8.0), 2 mM EDTA and 0.1% (w/v) SDS for 3 min. To increase the specificity of the probes, membranes were incubated in the same so lution for 3 min at exactly 59 °C. After incubation, they were rinsed twice in 2 x SSPE for 3 min to re move any remaining tétraméthylammonium chlo ride. Subsequently, the membranes were soaked in a reaction buffer containing 100 mM Tris-HCI (pH 7.2) and 150 mM NaCl (buffer I) for 5 min. then moved to buffer I plus 0.5% (w/v) blocking reagent (Boehringcr-Mannheim) for 30 min. The mem branes were reacted with 150 mU/ml alkaline-phos phatase-conjugated antidigoxigenin (BoehringcrMannheim) in buffer I for 30 min. then washed twice in buffer 1 for 10 min. Color development (30 min) was performed using a solution containing 100
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 8/12/2018 11:11:17 PM
Fig. 1. DNA typing of HLADQA1 genotypes with 10 SSO probes in the Japanese population. As a positive/negative control, DNA from HLA homozygous cell lines was used. P = Positive con trols as follows: SSO 1,9 = HOM2. 9005. DQA 1 *0101; SSO 2, 7 = BM16. 9038, DQA 1*0501; SSO 3, 6 = BMI4. 9033. DQA 1*0301; SSO 4 = WDV. 9062. DQA 1*0103; SSO 5. 10 = DBB, 9052, DQA 1*0201: SSO 8 = BM9. 9068. DQA 1*0401; N = negative controls as follows: SSO 3, 6 = BMI6: others BM14. Data arc expressed as name of cell line, workshop number and allele.
Tamaki/Yamamoto/Uchihi/Katsumata/Kondo/Mizuno/Kimura/Sasazuki
Tabic 2. Distribution of HLA-DQA1 genotypes in the Japanese population DQAI subtypes
Observed n
*01011*0101 *01011*0102 *01011*0103 *01011*0201 •01011*0301 *01011*0401 *01011*0501 *01011*0601 *01021*0102 *01021*0103 *01021*0201 *01021*0301 *01021*0401 *01021*0501 *01021*0601 *01031*0103 *01031*0201 *01031*0301 *01031*0401 *01031*0501 *01031*0601 *02011*0201 *02011*0301 *02011*0401 *02011*0501 *02011*0601 *03011*0301 *03011*0401 *03011*0501 *03011*0601 •04011*0401 *04011*0501 *04011*0601 *05011*0501 *05011*0601 *06011*0601 Total
Expected %
5 II 13 0 23 1 4 2 6 16 1 37 6 9 2 12 1 50 2 6 1 0
0 0 0 0 50 10 11 5 0
4 0 2 0
1.7 3.8 4.5 0.0
7.9 0.3 1.4 0.7 2.1 5.5 0.3 12.8 2.1 3.1 0.7 4.1 0.3 17.2 0.7 2.1 0.3 0.0 0.0
0.0 0.0
0.0 17.2 3.4 3.8 1.7 0.0 1.4 0.0
0.7
0
0.0 0.0
290
99.8
X2 = 20.2; d.f. = 28; 0.80 < p
© 1991 S.K arger A G . Basel 0001-5652/91/0414-0209 S 2.75 II
Frequency of HLA-DQA1 Alleles in the Japanese Population Keiji Tamakia, Toshimichi Yamamotoa, Rieko Uchihi", Yoshinao Katsumata3, Keiko Kondob, ShinichiMizunob, AkinoriKimurac, Takehiko Sasazukic a Department of Legal Medicine, Nagoya University School of Medicine, Nagoya; h Japanese Red Cross Aichi Blood Center, Seto-shi; c Department of Genetics, Medical Institute of Bioregulation. Kyushu University. Fukuoka. Japan
Key Words. DNA polymorphism • HLA-DQA1 locus • Polymerase chain reaction Abstract. One of the HLA class II genes, HLA-DQAi, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQAI gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQAI ge notype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the num bers expected under the Hardy-Weinberg equilibrium. The HLA-DQAI genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.
(PCR) procedure [1] allows the enzymatic amplification of a specific segment of hu The HLA class II molecules DR. DQ man genomic DNA and direct detection of and DP are highly polymorphic cell sur the allelic variation in the HLA-DQA lo face molecules which have an important cus [2, 3], The DQA genotypes of single function in the generation and regulation hairs can be detected using sequence-spe of the immune response. The elucidation cific oligonucleotide (SSO) probes [4], It of their polymorphisms is thus important has also been suggested that the allelic for understanding the mechanism of allo- variation in the HLA-DQA locus detected rccognition and the generation of autoim by SSO probes was greater than the poly mune diseases. These polymorphisms are morphisms defined by serological typing usually detected serologically using vari [ 21. ous antibodies. However, recent develop Recently, the WHO Nomenclature for ment of the polymerase chain reaction Factors of the HLA System Committee
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 8/12/2018 11:11:17 PM
Introduction
210
Tamaki/Yamamoto/Uchihi/Katsumata/Kondo/Mizuno/Kimura/Sasazuki
No.
DQA1 specificity
Sequence (5-3')
1 2
0101 0102, 0103, 0501 0101. 0102. 0201, 0301 0103 0201 0301 0501 0401. 0601 0101, 0102, 0401, 0501 0103, 0201. 0601
CACGTAGAACTCCTCATC
3 4 5 6 7 8 9 10
CACGTAGAACTGCTCATC CTCCTTCCTCTCCAGGTC GTCTCCTTCTTCTCCAGG CAGAGGCAACTTCCAGAC ATCTTCTAAATCTGCGGA TCAGACTGTTCAAGTTAT CGTAGAACTGCTCGTCTC TCATGGGTGTACTGGCCA TCATGGGTGAACTGGCCA
proposed the nomenclature of HLA al leles and loci defined by nucleotide or in ferred amino acid sequences [5]. Under the nomenclature, the HLA-DQA1 locus has 8 defined alleles as DQA1*0101, DQA 1*0102, DQA1*0103. DQA1*0201, DQA1*0301, DQA 1*0401, DQA1*0501 and DQA1*0601. However, the gene frequen cies of each allele of HLA-DQA1 have not been reported so far. The following report describes the gene frequencies of HLADQA1 in the Japanese population using a set of SSO probes. Furthermore, HLADQA1 genotypes were determined with aged bloodstains using the present method. Materials and Methods Extraction o f DNA from Blood Blood samples were obtained from 290 healthy Japanese and mixed with heparin in small test tubes. They were centrifuged and the huffy coat was col
lected. The huffy coat was rinsed twice with 2 vol of a solution containing 0.01 M Tris-HCI (pH 7.6). 0.005 M MgCI, and 0.01 M NaCl. then resuspended in one third volume of a solution containing 0.01 M TrisHCI (pH 7.6). 0.01 M EDTA (pH 8.0). 0.05 M NaCl. 0.5% (wlx) sodium dodecyl sulfate (SDS) and 200 gg/ml proteinase K (Sigma Chemical. St. Louis, Mo.. USA). Actual solvent volumes were dependent on the volume of blood used. The final suspension was incubated overnight at 42 °C. DNA in the specimens was extracted twice with phenol/chloroform (1:1) and once with chloroform, and was then precipitat ed with isopropanol. The precipitation was rinsed 3 times with 70% (w/v) ethanol, dried under vacuum, dissolved in a solution containing 0.01 M Tris-HCI (pH 7.4) and 0.001 M EDTA (pH 8.0) and stored at 4°C until required for use. The concentration of DNA was determined by measuring the optical den sity at 260 nm. DNA was extracted from bloodstains according to the protocol described by Gill et al. [6], PCR Amplification o f DNA DNA (1 gg) was amplified by PCR using the fol lowing primers: GH26 (5'-GTGCTGCAGGTGTAAACTTGTACCAG-3') and GH27 (5'-CACGGATCCGGTAGCAGCGGTAGAGTTG-3') |7|. Two units of Taq DNA polymerase (Perkin-ElmerCetus, Norwalk. Conn., USA) and 20 pmol of each primer were added to polymerase buffer (100 gl) containing 10 mM Tris-HCI (pH 8.4), 50 mM KCI. 2.5 mM MgC!,, gelatin (1(H) gg/ml) and 200 gAJ each of the four dcoxyribonucleoside 5'-triphosphatcs (dATP. dCTP. dGTP and TTP). 40 cycles of PCR were carried out using an automated device (DNA Thermal Cycler. Perkin-Elmer-Cetus), and each cy cle was performed for 2 min at 94. 55 and 72 °C. Af ter the last cycle, the samples were incubated for an additional 5 min at 72 °C. The amplified DNA was checked using a gel of 3% (w/v) NuSieve (FMC, Rockland, Me., USA) stained with ethidium bro mide. Preparation o f Labeled SSO Probes Ten SSO probes were devised as described by Fukui et al. [8] (table I) based on published HLADQA1 alignments [3, 9-15], They were synthesized by a 381A DNA Synthesizer (Applied Biosystems, Foster City, Calif.. USA). Following a conventional purification procedure, probe concentrations were determined by measurement of absorbance at
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 8/12/2018 11:11:17 PM
Table 1. SSO probes used in the present study
211
Frequency of HI.A-DQAI Alleles in Japanese
DQA1
SSO °1 •
«
•
• •
2 3 •
V
•
•
•
•
4 •
•
•
•
•
4
©
•
•
r
•
•
• •
5 • 6 • 7
•
8
•
9
•
10
•
260 nm. 30 pmol of each oligonucleotide were la beled with digoxigenin-ll-dUTP (2.5 nmol; Boehringer-Mannheim. Mannheim, FRG) in a labeling buffer [140 \iM sodium cacodylate, 30 mM Tris-FICI (pH 7.2). I mM CoCI,. 100 gM dithiothreitol] and 20 units of terminal transferase for 2 h at 37 °C. Hybridization and Detection o f HLA-DQA 1 Genotypes All the following procedures were performed at room temperature unless otherwise stated. A nylon membrane (Hybond N+. Amersham. Amersham, UK) of suitable size was completely soaked in 10 x SSPF for 10 min: I x SSPE is 0.15 M NaCl, 0.01 M so dium phosphate and 1 mW EDTA (pH 7.4). Ampli fied DNA (5 pi) was applied to a membrane in a Bio-Dot microfiltration apparatus (Bio-Rad. Rich mond. Calif.. USA). After drying completely, the membrane was soaked in a solution containing 0.4 M NaOH for 1 min and then in 10 x SSPE for 5 min, and dried completely again. Membranes were prcincubated in 6x SSPE, 0.1% (w/v) sodium N-lauroyl sarcosinate, 0.02% (w/v) SDS and 5 x Denhardt’s solution: 1 x Dcnhardt's solution is 0.02% (w/v) Ficoll 400 (Pharma
•
•
1
•
• •
o
•
•
• •
•
•
•
•
•
•
•
•
•
•
•
•
•
cia, Uppsala, Sweden), 0.02% (w/v) bovine serum al bumin (Fraction V, Wako Pure Chemical, Osaka. Ja pan) and 0.02% (w/v) polyvinylpyrrolidone, for 20 min at 42°C. Hybridization was performed in the same solution as prehybridization containing 2 nM SSO probes for 20 min at 42°C. After hybridization the membranes were washed for 5 min with 2x SSPE, then soaked in a solution containing 3.0 M tétraméthylammonium chloride, 50 mM Tris-HCI (pH 8.0), 2 mM EDTA and 0.1% (w/v) SDS for 3 min. To increase the specificity of the probes, membranes were incubated in the same so lution for 3 min at exactly 59 °C. After incubation, they were rinsed twice in 2 x SSPE for 3 min to re move any remaining tétraméthylammonium chlo ride. Subsequently, the membranes were soaked in a reaction buffer containing 100 mM Tris-HCI (pH 7.2) and 150 mM NaCl (buffer I) for 5 min. then moved to buffer I plus 0.5% (w/v) blocking reagent (Boehringcr-Mannheim) for 30 min. The mem branes were reacted with 150 mU/ml alkaline-phos phatase-conjugated antidigoxigenin (BoehringcrMannheim) in buffer I for 30 min. then washed twice in buffer 1 for 10 min. Color development (30 min) was performed using a solution containing 100
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 8/12/2018 11:11:17 PM
Fig. 1. DNA typing of HLADQA1 genotypes with 10 SSO probes in the Japanese population. As a positive/negative control, DNA from HLA homozygous cell lines was used. P = Positive con trols as follows: SSO 1,9 = HOM2. 9005. DQA 1 *0101; SSO 2, 7 = BM16. 9038, DQA 1*0501; SSO 3, 6 = BMI4. 9033. DQA 1*0301; SSO 4 = WDV. 9062. DQA 1*0103; SSO 5. 10 = DBB, 9052, DQA 1*0201: SSO 8 = BM9. 9068. DQA 1*0401; N = negative controls as follows: SSO 3, 6 = BMI6: others BM14. Data arc expressed as name of cell line, workshop number and allele.
Tamaki/Yamamoto/Uchihi/Katsumata/Kondo/Mizuno/Kimura/Sasazuki
Tabic 2. Distribution of HLA-DQA1 genotypes in the Japanese population DQAI subtypes
Observed n
*01011*0101 *01011*0102 *01011*0103 *01011*0201 •01011*0301 *01011*0401 *01011*0501 *01011*0601 *01021*0102 *01021*0103 *01021*0201 *01021*0301 *01021*0401 *01021*0501 *01021*0601 *01031*0103 *01031*0201 *01031*0301 *01031*0401 *01031*0501 *01031*0601 *02011*0201 *02011*0301 *02011*0401 *02011*0501 *02011*0601 *03011*0301 *03011*0401 *03011*0501 *03011*0601 •04011*0401 *04011*0501 *04011*0601 *05011*0501 *05011*0601 *06011*0601 Total
Expected %
5 II 13 0 23 1 4 2 6 16 1 37 6 9 2 12 1 50 2 6 1 0
0 0 0 0 50 10 11 5 0
4 0 2 0
1.7 3.8 4.5 0.0
7.9 0.3 1.4 0.7 2.1 5.5 0.3 12.8 2.1 3.1 0.7 4.1 0.3 17.2 0.7 2.1 0.3 0.0 0.0
0.0 0.0
0.0 17.2 3.4 3.8 1.7 0.0 1.4 0.0
0.7
0
0.0 0.0
290
99.8
X2 = 20.2; d.f. = 28; 0.80 < p
