Modified PCR-RFLP method for HLADPBl and -DQA1 genotyping M. Ota, T. Seki, N. Nomura, K. Sugimura, N. Mizuki, H. Fukushima, K. Tsuji, H. Inoko. Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping. Tissue Antigens 1991: 38: 6&71. Abstract: We previously developed a new technique for HLA class I1 genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class I1 alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthetmore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and MnlI can discriminate eight alleles of the DQAl gene. Similarly 19 alleles of the DPBl gene can be discriminated with Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.
Recently, genetic polymorphism in the HLA class
I1 region has been identified by the analysis of the polymerase chain reaction (PCR) products using sequence-specific oligonucletide (SSO) analysis (1-3). This PCR technique has been used extensively in the study of polymorphism, evolution (4) and forensic analysis (5), as well as in the analysis of genetic susceptibility to disease (6-9). The PCR method permits precise and direct analysis of allelic variations with as little as 1 ng of genomic DNA. We previously reported a rapid and simple method for HLA-DQA1, -DPB1, -DQB, and -DRB genotyping by digestion of PCR-amplified D N A with allele-specific restriction endonucleases (PCRRFLP method) (10-12). This PCR-RFLP method has some advantages over the PCR-SSO method which requires multiple SSO probes corresponding to the alleles, their labeling and washing temperatures usually with several stringencies (13-16). A non-radioactive "reverse dot blot" method has also been used for SSO typing (17), but the amount and length of oligonucleotide probes must be adjusted 60
Masao Ota, Takeshi Seki*, Nobuhiro Nomrra**, Kazuhitu Suglmura", NoLuAisa Mizuki", Hirofuml Rkushima, Kimiyoshl Tsuji** and Hidotashi Inoko" Department of Legal Medicine, Second Department of Internal Medicine, Shinshu University School of Medicine, Nagano, "Department of Transplantation, Tokai University School of Medicine, Kanagawa, Japan
Key words: polymerase chain reaction - restriction fragment length polymorphism - sequencespecific oligonucleotide - DM1 and DPBl alleles - genotype Received 14 November 1990, revised, accepted for publication 28 May 1991
and suitable conditions for hybridization determined, which is technically demanding. The PCRRFLP method is practical and conventional for genotyping, but small fragments or bands located close to each other on the polyacrylamide gels sometimes obstruct precise analysis and some heterozygotes cannot be discriminated from each other (18). In this study we searched for informative restriction enzymes which have a single recognition site in some alleles, but none in other alleles in the amplified segments of the HLA-DQA1 or -DPBl genes (19-26), making reading of the generated RFLP band patterns much simpler. The PCRamplified DNAs digested with these restriction endonucleases (ApaLI, HphI, BsaJI, FokI and MboII for DQA1, Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII for DPBl) were subjected to electrophoresis. DQAl and DPBl genotypes were determined just by checking whether the amplified DNAs are mainly digested or not. Heterozygotes could also be analyzed with
Modified PCR-RFLP for DP and DQ
DPB1*0301 n~~i*o4oi DPBl*0402
DPB~*O~O~ DPgl*OBOl DPBl*0801 DPBl*0901 DPB1:lOOl DPBl*llOl DPBlt1301 DPBltl8Ol DFBl*lSOI DPBltl401 DPBlt1501 DWl*lBOl DPBl.1701
Table 1. Correlation between cleavage patterns obtained by 9 restriction endonucleases and DPB1 alleles Restriction endonucleases ~~
OPE1 allele 0401 1801 = 0402' 0201 0202 0801 = 1601 0501 0301 0601 1101 0101 0901 = 1701 1001 1301 1901 1401 1501
~
______~
Group
Bsp12861
Fokl
Ddel
BSall
BssHll
Or131
Rsal
EcoNl
Avall
A
1 1 1 1 0 1
1 1 1 1 1 1
0
1 0 0 0 0
1 1 1 1
0
0 1 1 1 0 1
0 0
0
0
1 1 1 1 1 1 1 0 1
1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1
0
I 0
1 1 1 1 1 1 0 0 0 1
1 1 0
1
0 C D E F G H I J
K L M N
0 P
0 0
1 1
0 0 1 1
0 0 1 0 1 0 0 0 0 0 0
0 1 0 0
0 0 0 1 0
1
0 0 0 1 1 0 0 1
0
0 1 1 1 0
0 0 0
0
0
0
1 1
1
1 1 1 1 1 1
0 0
0 1 1 0 0 0 0 0 1 1
0: not cleaved, 1: cleaved. *: DPB1'1801 and 0402 can be discriminated by detecting RFLP bands digested with Rsal. A 147bp fragment is positive for DPB1'1801, a l77bp fragment positive for OPBl"0402 and heterozygote OPB1'1801/0402 has both fragments (147 and 177 bp fragments). ': DPB1'0801 and ,1601 can be discriminated by detecting RFLP bands after digestion with Fokl (116bp positive for 0801 and 59bp positive for 1601). I: DPB1'0901 and 1701 can be discriminated by detecting RFLP bands after digestion with Fokl (116bp positive for 0901 and 59bp positive for 1701) (see Tables 2, 4, 5 and 6).
61
Ota et al. Table 2. Fragments (bp) detected by digestion of the PCR-amplified DPBl genes by 9 different restriction endonucleases Restriction Endonucleases
DPB1 alleles
Bspl2861
Fokl
We1
BsJl
BssHll
Cfrl31
Rsal
EcoNl
Avall
0401 0402 0201 1801 0202 0801 0501 0301 0601 1101 0101 0901 1001 1301 1901 1401 1501 1601 1701
262.37 262,37 262,37 262,37 153,109,37 299 190,109 299 299 299 299 268,31 268,31 299 299 268,31 262,37 299 268,31
186,57,56 183,59,57 183,5937 183,5937 183,59,57 183,116 183,59,57 299 242,57 242,57 183,116 183,116 183,116 183,116 183,116 299 242,57 183,59,57 183,59,57
299 299 299 299 166,133 299 166,133 299 299 299 299 299 299 299 166,133 299 299 299 299
246.32,21 246.32,21 246,32,21 246,3221 246,32,21 246,32,21 246.32.21 278,21 278,21 278,21 246,32,21 278,21 278.21 278,21 246.32,21 278,21 246.32.21 246.32.21 278.21
188,111 299 299 299 299 299 299 299 299 181,111 188,111 299 299 188.1 11 299 299 188,111 299 299
258,40 258,40 258,40 258,40 258.40 299 299 194.1 05 194,105 194.1 05 299 299 299 299 299 194,105 194,64,40 299 299
177.1 22 177,122 177,122 147,122,30 177.1 22 177,122 177,122 269,30 269,30 147,122.30 122,78,69,30 299 177,122 122,78,69,30 177,122 299 122,78,69,30 177,122 299
269,30 269,30 174,95,30 299 174,95,30 174,95,30 269,30 201,98 201,95,3 201,98 299 204,95 204,95 201,98 174,95,30 201,98 201,98 174,95,30 204,95
299 299 299 299 299 299 299 194,105 194.1 05 194,105 299 299 299 299 299 194,105 194,105 299 299
the method. This improved PCR-RFLP method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method, and so is a good alternative to the PCRSSO method.
Material and Methods DNA samples
70 DNA samples used in this study were distributed for Southern blot analysis in the Tenth International Histocompatibility Workshop. They were isolated from EBV-transformed HLA homozygous B-lymphoblastoid cell lines which were used in the previous study (10, 11). Genomic DNAs from 100 healthy Japanese volunteers were isolated by phenol extraction of sodium dodecyl sulfate (SDS)lysed and proteinase K-treated cells, as described earlier (27). PCR amplification
Genomic DNA (1 pg) was amplified by the PCR procedure with 2.5 units of the Taq DNA polymerase (Perkin Elmer Cetus Inc.) (28). The reaction mixture was subjected to 30 cycles of 1 min at 94"C, 1 min at 62"C, and 2 min at 72°C by automated PCR thermal sequencer (Iwaki Glass Inc.). The second exon of the DQAl gene was amplified by using the PCR primers, GH26 and GH27 at 1 pM and the second exon of the DPBl gene was amplified by using the PCR primers, DPB 10IN and DPB201 at 1 pM (Fig. 1).
62
Digestion with restriction endonucleases
After amplification, aliquots (7 pl) of the reaction mixture were digested with restriction endonucleases (ApaLI, HphI, FokI, MboII, Bsp 12861, DdeI, Cfrl31, RsaI, EcoNI, and AvaII; 5 units) at 37°C for 3 h after addition of appropriate incubac tion buffer. When digested with BsaJI or BssHII, the reaction mixture was incubated at 60°C or 50°C for 1 h, respectively. ApaLI, HphI, BsaJI, FokI, MboII and MnlI were used for digestion of the amplified DQAl gene and Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII
Table 3. Correlation between cleavage patterns obtained by 5 restrictionendonucleases and WA1 alleles Restriction endonucleases wA1 allele
0101 0102 0103 0201 0301 0501 0401 0601
Group
ApaLl
Hphl
A
1
0
0
0
0
B C D E F G
1 0 0 0 0 0
1 1 0 0 0 1
0 0 0 1 1 1
0 1 0 0 1 1
1
Bsall
Fokl
Mboll
0 1 0 0 0
0: not cleaved, 1: cleaved. DQA1 0101 and 0102 can be discriminated using Mnll. A 40bp fragment is positive for 0101 and negative for 0102. On the other hand, a 23bp fragment is positive for 0102 and negative for 0101 (see Table 11 and Fig. 3).
Modified PCR-RFLP for DP and DQ Table 4. Patterns of polymorphic fragments detected with Rsal enzyme for discrimination of heterozygotes between DPB1'1801 or 0402 allele and the other DPBl alleles Combinations DPBl alleles
040111801 040110402 180111801 180110402 040210402 020111801 020110402 020211801 020210402 0801 =160111801' 0801 = 1601/0402' 050111801 050110402 030111801 030110402 060111801 060110402 110111801 110110402 010111801 010110402 0901 = 170111801' 0901 = 170110402" 100111801 100110402 130111801 130110402 190111801 190110402 140111801 140110402 150111801 1501/0402
269 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
177 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 0 1 0 1 1 1 0 1 1 ' 1 0 1 0 1
147
122
1 0 1 1 0 1 0 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 1 0
1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0
1: positive, 0: negative. 5 RFLP bands digested with kkl can discriminate DPB1*0801/1801 (positive for 116 bp) and DPB1'160110201 (negative for 116bp). I: RFLP bands digested with Fokl can discriminate DPB1'080110402 (positive for 116 bp) and DPB1'160110402 (negative for 116 bp). *: RFLP bands digested with Fokl can discriminate DPB1'090111801 (positive for 116 bp) and DPB1'170110201 (negative for 116 bp). I: RFLP bands digested with Fokl can discriminate OPB1'0901/0402 (positive for 116 bp) and DP61'1701/0402 (negative for 116 bp).
were used for digestion of the amplified DPBl gene. Complete digestion of restriction enzymes was confirmed by including positive control DNAs with the HLA alleles which have cleavage sites for respective enzymes in the PCR-amplified regions. Acrylamide gel electrophoresis
Samples of the restriction enzyme-cleaved amplified DNAs were usually subjected to electrophoresis in 12% polyacrylamide gels in a horizontal minigel apparatus (Mupid, Cosmo Bio Co. Ltd.). When digested with MnlI enzyme, 15% poly-
Table 5. Patterns of polymorphic fragments detected with Fokl enzyme for discrimination of heterozygotes between DPB1'0801 or 1601 allele and the other DPBl alleles Combinations DPBl alleles
040110801 040111601 1801 =0402/0801' 1801 = 040211601H 020110801 601 0201/I 020210801 020211601 080110801 080111601 160111601 050110801 050111601 030110801 030111601 0601/a801 060111601 110110801 110111601 D10110801 010111601 090110801 170110801* 090111601' 170111601 100110801 100111601 130110801 130117601 190110801 190111601 140110801 140111601 150110801 ' 150111601
FokUbP)
183
116
59
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 0
0 1 1 1 1 1 1 1 0 1 1 1 1 0 1 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1
1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 0
1: positive, 0: negative. *: RFLP bands digested with Rsal can discriminate DPB1'180110801 (positive for 147 bp) and DPB1*0402/0801 (negative for 147 bp). #: RFLP bands digested with Rsal can discriminate DPB1'18011 1601 (positive for 147bp) and DPB1'0402/1601 (negative for 147bp). ': 170110801 and 090111601 give the same RFLP band pattern and cannot be discriminated from each other.
acrylamide gel was used. Cleavage or no cleavage of amplified fragments was detected by staining with ethidium bromide.
Results Analysis of nucleotide sequences of PCR-amplified regions in the DPBl and DQA1 genes for allele-specific restriction
sites Fig. 1 summarizes the allelic variations in the 299bp PCR-amplified region of the second exon in the DPBl genes reported by published papers (21-26), where polymorphism in the DPB locus is 63
Ota et al.
primarily localized. These DNA sequences were used to search for restriction endonucleases which have a single cleavage site in some alleles (indicated by 1 in Table 1) but none in other alleles (indicated by 0 in Table 1) in the amplified region by computer analysis and 9 different restriction endonucleases, Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII, were selected for digestion' to detect allele-specificcleavage after PCR-amplification of the DPBl alleles (Table 1). Table 2 shows the sizes of the fragments detected by digestion of these restriction enzymes. Here, eight DPBl alleles (DPBl*0201, *0202,.*0401, *0402, *0501, *0801, Table 6. Patterns of polymorphic fragments detected with Fokl enzyme for discrimination of heterozygotes between DPB1'0901 or 1701 allele and the other OPBl alleles Combinations DPBl alleles 040110901 0401/1701 1801 = 0402/0901* 1801 = 0402/1 7Olx 020110901 020111701 020210901 020211701 080110901 160110901 080111701* 160111701 050110901 050111701 030110901 030111701 060110901 060111701 1 10110901 1101/1701 0101/0901 010111701 090110901 090111701 1701/1701 100110901 100111701 130110901 1301I1701 190110901 190111701 140110901 140111701 150110901 150111701
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 0
1 0
1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 0
0 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 0 1 0 1 0 1 0 1 1 0
1 0 1 0 1 0 1 0 1
1: positive, 0: negative. *: RFLP bands digested with Rsal can discriminate DPB1'180110901 (positive for 147bp) and DP61*0402/0901 (negative for 147bp). #: RFLP bands digested with Rsal can discriminate DP61'180111701 (positive for 147bp. negative for 177bp) and DPB1'040211701 (negative for 147bp, positive for 177bp). *: 1601/0901 and 0801/1701 give the same RFLP band pattern and cannot be discriminated from each other as described in the foodnote of Table 5.
64
*1901 and *1601) have a cleavage site for EcoNI at the 30th nucleotide position from the 5' terminal and so, in the case of EcoNI, 269bp fragmentpositive alleles were also indicated by 0 for simplification in Table 1. For the same reason, 278bp BsaJI fragment-positive alleles were also indicated by 0 in Table 1. As shown in Table 1, these 9 different enzymes predict discrimination of 16 of 19 DPBl alleles, because the DPB1*1801 and DPB1*0402, DPB1*0801 and DPBl*1601, and DPB1*0901 and DPB 1* 1701 alleles give the same cleavage pattern. However, these three pairs of alleles can be discriminated by RFLP band patterns obtained with digestion of RsaI or FokI enzyme (Table 2, see also Tables 4 to 6). Digestion with RsaI restriction endonuclease gave a 147bp fragment for the DPB1*1801 and a 177bp fragment for the DPB1*0402 allele, and both fragments (147bp and 177bp) for DPBl* 1801/0402 heterozygote. Digestion with FokI restriction endonuclease gave a 116bp fragment for the DPB1*0801, a 59bp fragment for the DPB 1* 1601 allele and both fragments (1 16bp and 59bp) for DPB1*0801/1601 heterozygote. Further, digestion with FokI restriction endonuclease gave a 116bp fragment for the DPB1*0901, a 59bp fragment for the DPBl*1701 allele and both fragments (1 16bp and 59bp) for DPBl*O901/1701 heterozygote. In a similar way, the allelic variations in the 242bp PCR-amplified region of the DQAl genes were used to search for restriction endonucleases specific for each of eight DQAl alleles by computer analysis, and 5 different restriction endonucleases, ApaLI, HphI, BsaJI, FokI, and MboII, were selected to detect allele-specific cleavage after PCRamplification (Table 3). As shown in Table 3, these 5 restriction enzymes are expected to allow discrimination of all but two (seven out of eight) DQAl alleles because the DQA1*0101 and DQA1*0102 alleles give the same cleavage pattern. This problem was overcome by digestion with MnlI restriction endonuclease which distinguished the DQA1*0101 allele from the DQA1*0102 allele on the basis of the RFLP band patterns (see Fig. 3 and Table 11). A 40bp fragment is positive for the DQA1*0102 allele and negative for the DQAl*0101 allele. On the other hand, a 23bp fragment is positive for DQAl*O101 but negative for DQA1*0102. Digestion patterns by allele-specific restriction endonucleases in the amplified DPBl and DQAl genes from homozygous and heterozygous individuals
In order to test the method developed above, genomic DNAs from 70 HLA-homozygous B-cell lines with different DP and DQ specificities and from
Modified PCR-RFLP for DP and DQ Table 7. Cleavage patterns obtained by restriction enzymes in DPBl heterozygotes
Restriction Endonucleases
Combinations of DPBl alleles
Groups
0401I1801 =0402' 0401I0201 040110202 0401I0801 = 1601' 0401/0501 0401I0301 040110601 0401I1101 040110101 040110901= 1701 0401I1001 0401I1301 0401I1901 0401I1401 0401I1501 '1 801 = 0402/0201 '1 801 = 0402I0202 '1 801 = 0402/0801= 1601' '1801 = 0402I0501 7801 = 0402I0301 '1 801 = 0402I0601 '1 801 = 040211101 '1 801 =0402/0101 '1 801 = 0402/0901= 1701 '1 801 = 0402I1001 '1 801 = 0402I1301 '1 801 = 0402I1901 "1 801 = 0402I1401 '1 801 = 0402I1501 0201/0202 0201I0801 0201/0501 0201I0301 020110601 0201I1101 0201/0101 0201/0901= 170lX 0201I1001 0201I1 301 0201I1901 401 0201/I 0201I1501 0202/0801= 1601' 0202/0501 0202I0301 0202I0601 0202/1101 0202/010? .0202/0901= 1701 0202/1001 0202I1301 0202/1901 0202/1401 0202I1501 %80l= 1601/0501 WE01 = 1601I0301 %SO1 =160110601 '0801 = 1601I1101 %a01 = 1601I0101 '0801 =160110901=1701I
A/B A/C A/D A/E"
NF A/G A/n2) All A/J A/K AIL AIM A/N3) NO
A/P BIG B/O
BIE BIF BIG BIH B/l2' B/J BIK BIL B/M BIN BIO BIP
c/o
CIE CIF CIG CIH CII CIJ" C/K CIL CIM CIN"
c/o CIP
D/P' DIF DIG OIH D/I 01~3)
DIK D/L
DIM DIN
DIO DIP UF UG UH
UI UJ
OK
Bsp12861
Fokl
Ddel
BSdl
BssHll
Cfrl31
Ral
EWNl
Avail
1 1 1 2 1 2 2 2 2 1
-1 1 1
0 0 2 0 2 0 0 0
1
2 2 2 2 2 2 2 1 1 2 2 1 2 2 1 0 0 0 0 0 0 2 2 0 0 2 0 0 2 0 0 0 0 0 2 2 0 0 2
1
1
1 1 2 2 1 1 1 2 2 2 2 2 1 1 1 1 2 2 1 1 1 2 2 2 2 2 1 1 1 2 2 1 1 1 2 2 2 2 2 1 1 2 2 1 1 1 2 2 2 2 2 1 1 0 2 2 2 0 0
1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 2
0 2 2
0 0
1
2 2 1 1 1 1 2 1 2 2 2 2 1 1 2 2 1 1 1 2 1 2 2 2 2 1 1 2 2 1 1 2 1 2 2 2 2 1 1 2 2 1 1 2 0 0 0 0 2
1 1 2 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 2 1 1 2 1 1 1 1
0 0 0 0 2 0 0 0 2 0 2 0 0 0
0 0 0 0 2 0 0 2 0 2 0 0 0
0 0 0 0 2 0 0 2 1 2 2 2 2 2 2 2 1 2 2 2 0 0 0
0 0
1 1 1 1 2 2 2 1 2 2 2 1 2 1 1 1 1 1 2 2 2 1 2 2 2 1 2 1 1 1 1 2 2 2 1 2 2 2 1 2 1 1 1 2 2 2 1 2 2 2 1 2 1 1 2 2 2 1 2
0
0 2 0 0 0 0 2 2 0 0 2 0 0 2 0 0 0 2 2 0
2 0 2 2 2 0 2 2 2 2 2 2 2 2 2 0 2 2 2 0 2 2 2 2 2 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 2 1 1 1 2 1
0 0 0 2 2 2 0 0 0 0 0 2 2 0 0 0 0 2 2 2 0 0 0 0 0 2
2 0 0 0 2 2 2 0 0 0
0 0 2 2 0 0 2 2
2 0 0 0 0 0 2 2 0 2 2 2 0 0 contd.
Ota et al. Table 7 (contd.) Combinations of
Restriction Endonucleases
DPBl alleles
Groups
'0801 = 1601I1001 s0801= 1601/1301 $0801= 1601I1901 '0801 = 1601I1 401 $0801= 1601I1 501 050110301 0501/0601 0501I1 101 0501I0101 050110901 = 1701# 0501I1001 0501/1301 0501I1901 0501/1401 0501I1501 0301I0601 0301/1101 0301/0101 0301/0901=1701# 0301/1001 0301I1 301 0301/1901 0301I1 401 0301I1501 060111101 0601I0101 0601I0901 0601/1001 0601I1 301 0601I1901 0601I1401 0601I1 501 1101/0101 1101/0901=17Olx 1 101I1001 110111301 1 101I1901 1101/1401 1101/1501 0101/0901=1701# 0101I1 001 010111301 01 01I1 901 010111401 0101Il501 f0901= 1701I1 001 x0901= 1701I1301 %901= 1701I1 901 #0901= 1701I1 401 80901=1701/1501 1001I1301 1001I1 901 1001I1 401 1001I1 501 1301/1901 1301I1 401 1301I1501 1 901I1401 1901I1501 1401I1501
EIL EIM EIN
fY0 UP FIG FIH FII FIJ FIK FIL FIM F/N
fikl
Ddd
BMl
BSSHll
Or131
Rsal
2 0 0 2 2 2 2 2 2 1
1 1 1 2 1 2 1 1 1 1 1 1 1 2 1 2
0 0 2 0 0 2 2 2 2 2
2 2
0 2 0 0 2 0 0 2 2 0 0 2 0 0 2 0 2 2 0 0 2 0 0 2 2 2 0 0 2 0 0 2 1 2 2 1 2 2 1 2 2 1 2 2 1 0 2 0 0
0 0 0 2 2 2 2 2 0 0 0 0 0 2 2 1 1 2 2 2 2 2 1 1 1 2 2 2 2 2 1 1 2 2 2 2 2 1 1 0 0 0 0 2 2 0 0 0 2 2 0 0 2 2 0 2 2 2 2 1
1 1 1 2 1 1 1 1 1 2 1
1
2 2 1 1 0 0
F/O
F/P GIH G/I GIJ GIK GIL GIM GIN GI0
G/P H/I HIJ HIK HIL HIM HIN HI0
H/P IIJ IIK IIL IIM IIN 110
IIP JIK JIL JIM JIN JIO JIP WL WM WN WO UM UN
uo
2
2 2 0 0 2 2 0 0 2 2 0 0 2 2 0 2 2 0 0 2 2 2 2 0 0 2 2 1 2 2 1
2
0
2
0 0
1 1 0 2 2
UP MIN MI0
MIP
2
NIO NIP
2 1
OIP
2
2 2 1 2 2 0 0 0
0
1 2 2
WP
~-
Bsp12861
2 2 0 2 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 2 1 1 1 2 1 1 2 1 2 1 2
2
0 0 0 0 0 0 0 2 0 0
0 0 0 0 2 0 0 0 0 0 2 0 0 0 0 2 0 0 0 2 0 0 2 0 0 2 2
0
1 2 1 2 2 2 1 2 2 2 1 2 1 0 0 2 0 0 0 2 0 2 0 2 0 0 0 2 0 2 2 0 0 0 2 0 2
2 2 2 1 2 1
0
0 2
0 2
2
0 2 0
2
2 2 0 2 2 1 2
0 0 2
2 2 1 0 2 2
1
1 2 1 1 1 1 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 2 1 1 1 2 1 2 1 1 1 2 1 2 2
2 0 2 1
1
2 1 1 2 1
2 1 2
EcoNl
Avafl
1
0 0 0 2 2 2
1 1 1 1 2 2 2 0 2 2 2 2 2 2 1 1 2 1
1 1 1 1 1 1 2 1 1 1 1 1 1
2 1 1 1 1 1
1 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 2 0 0 0 0 0 2 2 1 1 2 2 2 2 2 1 1 1 2 2 2 2 2 1 1
2 2 2 2 2 1 1 0 0 0 0 2 2 ' 0
0 0 2 2 0 0 2 2 0 2 2 2 2 1
~
0: not cleaved, 1: cleaved, 2: both for heterozygote. Following four combinations"-" give the same cleaving RFLP patterns obtained with 9 restriction endonucleases but can be distinguished by reexamination of RFLP band patterns as shown in Table 8. AUCJ'' can be discriminated by RFLP band patterns obtained by Fokl and Rsall. AWBI') can be discriminated by RFLP band patterns obtained by Rsal. ANIDJ3) can be discriminated by RRP band patterns obtained by Bspl2861. CNIDE') can be discriminated by RFLP band patterns obtained by Bspl2861 and Fokl. *: DP61'1801 and 0402 can be discriminated by detecting RFLP bands obtained by Rsal (Table 4). I: DPB1*0801 and 1601 can be discriminated by detecting RFLP bands obtained by Fokl (Table 5). #: OP61*0901 and 7701 can be discriminated by detecting RFLP bands obtained by Fokl (Table 6). Only one combination of 0801I1 701 and 0901I1 601 cannot be discriminated by any restriction enzymes so far examined.
66
Modified PCR-RFLP for DP and DQ Table 8. RFLP patterns detected with Fokl, Rsal and Bsp12861 restriction endonucleases Combinations of DPBl alleles
groups
0401/0801 0401/1601 0201/0101
AE AE
CJ
Fokl 183
116
122
78
1 1 1
1 0 1
1 1 1
0 0
Combinations of DPBl alleles
groups
0401/0601 1801/1101 0402/1101
AH El 81
groups
0401/1901 0202/0101
AN
269
177
122
1 0 0
1 0 1
1 1 1
Bspl2861 262
153
1 0
0 1
DJ
Combinations of DPBl alleles
groups
0202/0801 0202/1601 0201/1901
DE DE CN
1
Rsal
Combinations of DPBl alleles
Rsal
Bspl2861
Fokl
262
153
116
59
0 0 1
1 1 0
1 0
1 1
0: negative, 1: positive.
100 healthy Japanese individuals were subjected to amplification of the DQAl and DPBl genes, followed by digestion with the restriction endonucleases described and electrophoresis in 12% polyacrylamide gels. HLA-DPB1
16 of 19 DPBl alleles were found to be discriminated on the basis of the cleavage patterns with 9 different endonucleases using 70 HLA homozygous B-cell lines as expected from Table 1, although no B-cell lines with the 1101, 1401, 1801 or 1901 allele are available. Three pairs of indistinguishable alleles DPBl*1801 and DPBl*0402, DPB1*0801 and 1601 and DPB1*0901 and 1701 can be discriminated by RFLP bands detected with RsaI or FokI restriction endonuclease no matter what allele is combined with these alleles, as mentioned above (Tables 4,5 and 6). 16 standard DPBl PCR-RFLP patterns give 120 heterozygous combinations (Table 7). Note that heterozygotes carry both cleaved and uncleaved amplified bands, indicated by 2 here. Four pairs out of these combinations cannot be distinguished by cleavage patterns with 9 restriction enzymes (DPB1*0401/
Figure 2. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DPBl genes obtained from DNAs of 4 normal individuals after digestion with 9 restriction enzymes. Determination of their genotypes was based on Tables 1,4,5,6,7 and 8. The size of the amplified region of the DPBl exon 2 is 299 bp as indicated. Two EcoNI-digested samples, DPB1*0501/0501 and 0402/0501 gave a little bit lower band (269bp), but were indicated by 0 for the reason described in the text.
0801 = 1601 and 0201/0101,0401/0601 and 1801= 0402/ 1101,0401/ 1901 and 0202/0101,0202/0801= 1601 and 0201/1901). However, these four pairs can be discriminated by reexamination of RFLP band patterns obtained after treatment of FokI, RsaI or Bsp1286I enzyme (Table 8). DPBl*0401/ 0801, 0401/1601 and 0201/0101 heterozygous alleles can be discriminated by the presence of the 116bp fragment digested with FokI enzyme and the 78bp fragment digested with RsaI enzyme. Discrimination of heterozygous DPB 1*0401/0601, 1801/1101 and 0402/1101 genotypes can be done by examining the presence of the 269bp or 177bp fragment digested with RsaI enzyme. DPB1*0401/ 1901 and DPB1*0202/0101 can be distinguished by examining the presence of the 262bp or 153bp fragment after digestion with Bsp1286I enzyme. Lastly, heterozygotes between DPBl*O201/0801, 0202/1601 and 0201/1901 can be discriminated by examining the 262bp or 153bp fragment with Bsp1286I and the 116bp fragment with FokI enzyme. Heterozygotes between DPBl * I80 1 or *0402 and the other DPBl alleles can be discrimi67
Ota et al. Table 9. HLA-OPE1 and DQAl allele frequencies in Japanese DPBl
n
Freq(%)
G.F.(%)
WA1
n
0101 0201 0202 0301 0401 0502 0501 0601 0801 0901 1001 1101 1301 1401 1501 1601 1701 1801 1901
0 32 9 7 9 23 57 1 0 19 0 0 3 2 0 1 0 0 0
0 32 9 7 9 23 57 1 0 19 0 0 3 2 0 1 0 0 0
0.0 17.5 4.6 3.6 4.6 12.3 34.4 0.5 0.0 10.0 0.0 0.0 1.5 1.o 0.0 0.5 0.0 0.0 0.0
0101 0102 0103 0201 0301 0401 0501 0601
22 29 40 1 76 1 14 3
~~
Table 10. Cleavage patterns obtained by restriction enzymes in W 1 heterozygotes
heq(%) G.F.(%) 22 29 40 1 76 1 14 3
11.7 15.7 22.5 0.5 51.O 0.5 7.3 1.5
~
Total number examined is 100 G.F.: Gene Frequency, G.F was estimated according to the Beinstein formula.
G.F.=l-(l-freqE
nated by detecting RFLP bands after digestion of RsaI enzyme (Table 4) and heterozygotes between DPB1*0801 or *1601, or DPB1*0901 or *I701 and the other DPBl alleles can be discriminated by checking RFLP fragments detected with FokI enzyme (Tables 5 and 6). In this way, all of 189 homozygous and heterozygous combinations except for DPBl*1601/0901 and DPB1*0801/1701 can be clearly defined. DPBl*1601/0901 and DPB1*0801/1701 give the same RFLP pattern and cannot be discriminated from each other by any restriction enzymes so far available. In order to assess the possibility of DPBl genotyping on the basis of the cleavage patterns predicted by the combination of two standard patterns of DPBl homozygotes as shown in Table 1, genomic DNAs from 100 normal, randomly selected Japanese individuals were analyzed (Fig. 2). RFLP patterns obtained with 9 restriction endonucleases were matched with one of the patterns given in Tables 1, 4, 5, 6, 7 and 8, and all of their DPBl genotypes could be distinctively defined, and their gene frequencies are shown in Table 9. HLA-DQA1
Seven of eight DQAl alleles were found to be discriminated on the basis of the cleavage patterns with 5 restriction enzymes using 70 HLA homozygous B-cell lines as expected from Table 3. In this improved PCR-RFLP method, seven DQA PCR-RFLP patterns (Table 3) can be combined in 68
Combinations of
Restriction endonucleases
DQA1alleles
Groups
0101 = 0102/01OY 0101 = 0102/0201' 0101 = 010U0301' 0101 = 0102/0501* 0101 = 010210401' 0101 = 0102/0601' 0103/0201 0103/0301 0103/0501 0103/0401 0103/0601 0201/0301 020110501 0201/0401 020110601 0301I0501 0301I0401 0301I0601 0501/0401 0501I0601 0401/0601
A/B AJC AID
AJE AIF
AJG B/C BID BIE B/F BIG C/D
CIE CIF C/G
D/E OIF DIG
UF WG FIG
ApaLl
Hphl
Bsall
Fokl
Mboll
1 2 2 2 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 0 0
2 2 0 0 0 2 1 2 2 2 1 2 2 2 1 0 0 2 0 2 2
0 0 0 2 2 2 0 0 2 2 2 0 2 2 2 2 2 2 1 1 1
0 2 0 0 2 2 2 0 0 2 2 2 2 1 1 0 2 2 2 2 1
2 0 2 0 0 0 2 1 2 2 2 2 0 0 0 2 2 2 0 0 0
0: not cleaved, 1: cleaved, 2 both for hetrozygote. ': DQA1 0101 and 0102 can be discriminated by using Mnll enzyme (see Table 11).
28 homozygous and heterozygous ways (Table 10). All the 28 combinations can be distinguished from each other. If Mnll restriction endonuclease is included, the DQA 1*O 101 and DQA 1*O 102 alleles can also be distinguished (Table 11, Fig. 3) and all
Table 11. Patterns of polymorphic fragments detected with Mnll enzyme for discrimination of homozygotes or heterozygotes including MA1'01 01 or 0102 alleles Combinations of
Mnll(bp)
wA1 alleles 0101 0101 0102 0103 0201 0301 0501 0401 0601 0: negative, 1: positive.
0101 0102 0102 0101 0102 0101 0102 0101 0192 0101 0102 0101 0102 0101 0102
23
40
1 1 0 1 0 1 1 1 1 1 0 1 0 1 0
0 1 1 0 1 0 1 0 1 1 1 1 1 1 1
Modified PCR-RFLP for DP and DQ
of 36 homozygous and heterozygous combinations can be unequivocally discriminated. 20 cell lines belonging to A group (DQA1*0101 or DQA1*0102) in our previous study (10) could be further characterized by using MnlI and classified into DQAl *O 101/O101, DQA 1*0101/0102, or DQA1*0102/0102 genotype (Fig. 3, DQA1*0101/ 0102 is not shown). Their genotypes were found to correlate well with the Dw specificity. Namely, the DQA1*0101 allele is associated with the Dwl, Dw9 or DB2 specificity and DQAl*0102 allele is associated with the Dw2, Dw21 or Dw19 specificity. Genomic DNAs from 100 healthy Japanese individuals are amplified and typed for DQAl genotype by this modified PCR-RFLP method (Fig. 4). All of their DQAl genotypes and gene frequencies could be determined unequivocally (Table 9). Discussion
The modified PCR-RFLP method presented here which depends mainly on cleavage or no cleavage of the amplified segment by informative restriction enzymes provides a simpler and more practical
Figure 3. Patterns of polymorphic restriction fragments obtained with digestion of the PCR-amplified DQA1*0101 and 0102 genes by MnlI enzyme. DNA samples were electrophoresed in a 15% polyacrylamide gel.
technique than our previous PCR-RFLP method and can identify HLA alleles of individuals homozygous or heterozygous at the DQAl and DPBl loci. Flow charts for DQAl and DPBl genotyping by this method are illustrated in Fig. 5. All the possible 36 and 189 combinations of DQAl and DPB 1, respectively, including homozygotes and heterozygotes, can be unequivocally discriminated with the restriction endonucleases selected here with only one exception, DPB1*1601/0901 and *0801/1701 which cannot be distinguished even by the PCR-SSO technique. This indistinguishable combination can be discriminated only by using allele-specific primers for the DPB 1* 1601 or *0801 allele. All of the restriction enzymes used here are commercially available, but some of them are expensive at present (e.g. AvaII and MnlI). Such enzymes can be replaced by other cheaper restriction enzymes as described in the previous paper (10, 11) or omitted depending on the typing sensitivity to be required. One major problem of the PCR-RFLP technique is incomplete or partial digestion of the PCR products by restriction enzymes, which will obscure definite assignment of HLA alleles. As included in any other typing methods, this problem can be
Figure 4 . Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DQAl genes obtained from DNAs of 6 normal individuals after digestion with 6 restriction enzymes. Determination of their genotypes was based on Tables 3, 10 and 11. The size of the amplified region of the DQAl exon 2 is 242 bp as indicated. DQA1*0201, 0401, 0501 and 0601 alleles have a 3bp deletion in the PCR-amplified exon 2 region and so some heterozygotes (e.g. DQAl*OlO2/0601) give two PCR amplified bands with different sizes detected when there are no cleavage sites of either allele for the restriction enzymes used (e.g. B: MboII).
69
Ota et al.
0:z Tables 1.4.5.6
It not
\
/
It not
\
/
DPBl G s n o t Y p e
Figure 5 . Flow chart of the modified PCR-RFLP method for determining genotypes of DPBl and DQAl. X: arbitrary allele.
overcome just by preparation of positive control DNAs for ensuring that the digestion is complete. Further, PCR primers incorporating recognition sites for restriction enzymes used here will be more useful as internal controls. The DQAl and DPBl alleles of 70 HLA-homozygous B-cell lines used in the Tenth International Histocompatibility Workshop defined by this PCR-RFLP method were found to be in complete agreement with those defined by the PCR-SSO method (N. Mizuki et al., unpublished, A. Kimura, unpublished). Thus, the PCR-RFLP method provides a useful, reproducible and accurate genotyping technique for HLA alleles. These genotyping analyses can be easily managed with a personal computer program. This modified PCR-RFLP method eliminating the need for radioactive or non-radioactive labeling of multiple SSO probes is a good alternative to the PCR-SSO method and can be substituted for the conventional serological and cellular techniques. This modified PCR-RFLP technique has also been successfully extended and applied to DQBl and DRBl genotyping for HLA homozygous or heterozygous individuals in larger trials (N. Nomura et al., in press, M. Ota et al., submitted). Acknowledgments
We thank Dr. J. Trowsdale of Imperial Cancer Research Fund for critical reading of the manuscript and Dr. J. H. Lee of One Lamda for giving important information of restriction enzymes. This work was supported by a grant-in-aid for scientific research in 1990 from the Japanese Ministry of Education. References 1. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA.
70
Analysis of enzymatically amplified f3-globin and HLADQa DNA with allele-specificoligonucleotide probes. Nature 1986: 324: 163-6. 2. Bugawan TL, Saiki RK, Levenson CH, Watson RM, Erlich HA. The use of non-radioactive oligonucleotide probes to analyze enzymatically amplified DNA for prenatal diagnosis and forensic HLA typing. Biotechnology 1988: 6: 943-7. 3. Wu DY, Ugozzoli L, Pal BK, Wallance RB. Allele-specific enzymatic amplification of P-globin genomic DNA for diagnosis of sickle cell anemia. Proc Nail AcadSci USA 1989: 8 6 2757-60. 4. Paabo S. Ancient D N A extraction, characterization, molecular cloning, and enzymatic amplification. Proc Nut1 Acad Sci USA 1989: 86: 1939-43. 5. Higuchi R, Beroldingen CH, Sensabaugh GF, Erlich HA. DNA typing from single hairs. Nature 1988 332 5 4 M . 6. Greenberg SJ, Ehrlich GD, Abbott MA, Hurwitz BJ, Waldmann JA, Poiesz BJ. Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification. Proc Natl Acad Sci USA 1989: 8 6 2878-82. 7. Trucco G, Fritsch R, Giorda R, Trucco M. Rapid detection of IDDM susceptibility with HLA-DQj3-allelesas markers. Diabetes 1989: 38: 1617-22. 8. Todd JA, Bell JI, McDevitt HO. HLA-DQP gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 1987: 329 599-604. 9. Scharf SJ, Friedmann A, Brautbar C, et al. HLA class I1 allelic variation and susceptibility to pemphigus vulgaris. Proc Natl Acad Sci USA 1988: 8 5 3504-8. 10. Maeda M, Murayama H, Ishi H, Ota M, Tsuji K, Inoko H. A simple and rapid method for HLA-DQAl genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases. Tissue Antigens 1989: 34: 290-8. 11. Maeda M, Uryu N, Murayama N, et al. A simple and rapid method for HLA-DP genotyping by digestion of PCRamplified DNA with allele-specific restriction endonucleases. Human Immunology 1990: 27: 111-21. 12. Uryu N, Maeda M, Ota M, Tsuji K, Inoko H. A simple and rapid method for HLA-DRB and -DQB typing by digestion of PCR-amplified DNA with allele specificrestriction endonucleases. Tissue Antigens 1990: 35 20-3 1. 13. Horn GT, Bugawan TL, Long CM, Erlich HA. Allelic sequence variation of the HLA-DQ loci: relationship to serology and to insulin-dependent diabetes susceptibility. Proc Natl Acad Sci USA 1988: 85: 6012-6. 14. Eliaou JF, Humbert M, Balaguer P, et al. A method of
Modified PCR-RFLP for DP and DQ HLA class I1 typing using non-radioactive labelled oligonucleotides. Tissue Antigens 1989: 33: 471-85. 15. Obata F, Ito I, Kaneko T, et al. Oligonucleotide-genotyping as a method of detecting the HLA-DR2 (DRwlS)-Dw2,DR~(DRw~S)-DW~~,-DR~-DW~~, and -DR4-DKT2” haplotypes in the Japanese population. Tissue Antigens 1989: 33: 55CL8. 16. Bugawan TL, Begovich AB, Erlich HA. Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specidicoligonucleotide probes. Immunogenetics 1990: 32: 23141. 17. Saiki PK, Walsch PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequencespecific oligonucleotide probes. Proc NatZ Acad Sci USA 1989: 8 6 623W. 18. Inoko H. PCR-RFLP method holds great promise of complete HLA class I1 genotyping. Tissue Antigens 1990: 36: 88-92. 19. Todd JA, Bell JI, McDevitt HO. HLA-DQP gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 1987: 329: 599604. 20. Moriuchi J, Moriuchi T, Silver J. Nucleotide sequence of an HLA-DQ chain derived from a DRw9 cell line: genetic and evolutionary implications. Proc Natl Acad Sci USA 1985: 82: 3240-4. 21. Bugawan TL, Horn GT, Long CM, et al. Analysis of HLADP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP-a and DP-p loci. J Immun01 1988: 141 4024-30. 22. Gustafsson K, Widmark E, Jonson AK, et al. Class I1 genes of the human major histocompatibility complex. J Bio Chem 1987 262: 8767-86.
23. Lair B, Alber C, Yu WY, Watts R, Bahl M, Karr RW. A newly characterized HLA-DPP chain allele. Evidence for DPP heterogeneity within the DPw4 specificity. J Immunol 1988: 141: 1353-7. 24. Trowsdale J, Kelly A, Lee J, Carson S, Austin P, Travers P. Linkage map of two HLA-SBP and two HLA-SBa related genes: an intron in one of the SB genes contains a processed pseudogene. Cell 1984: 38: 241-9. 25. Kelly A, Trowsdale J. Complete nucleotide sequence of a functional HLA-DPP gene and the region between the HLA-DPP1 and DPal genes: comparison of the 5’ ends of HLA class I1 genes. NucZ Acids Res 1985: 13: 1607-21. 26. Begovich AB, Bugawan TL, Nepom BS, Klitz W,Nepom GT, Erlich HA. HLA-DPP allele is associated with pauciarticular juvenile rheumatoid arthritis but not adult rheumatoid arthritis. Proc Natl Acad Sci USA 1989: 86: 9489-93. 27. Inoko H, Ando A, Ito M, Tsuji K. Southern hybridization analysis of DNA polymorphism in the HLA-D region. Human Immunol 1986: 16: 304-13. 28. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988: 239: 487-91. Address: Dr. Masao Ota Department of Legal Medicine Shinshu University School of Medicine 3-1-1 Asahi, Matsumoto Nagano 390 Japan Tel. 0263-35-4600, ext. 5217 Fax: 0263-33-6458
71
Recently, genetic polymorphism in the HLA class
I1 region has been identified by the analysis of the polymerase chain reaction (PCR) products using sequence-specific oligonucletide (SSO) analysis (1-3). This PCR technique has been used extensively in the study of polymorphism, evolution (4) and forensic analysis (5), as well as in the analysis of genetic susceptibility to disease (6-9). The PCR method permits precise and direct analysis of allelic variations with as little as 1 ng of genomic DNA. We previously reported a rapid and simple method for HLA-DQA1, -DPB1, -DQB, and -DRB genotyping by digestion of PCR-amplified D N A with allele-specific restriction endonucleases (PCRRFLP method) (10-12). This PCR-RFLP method has some advantages over the PCR-SSO method which requires multiple SSO probes corresponding to the alleles, their labeling and washing temperatures usually with several stringencies (13-16). A non-radioactive "reverse dot blot" method has also been used for SSO typing (17), but the amount and length of oligonucleotide probes must be adjusted 60
Masao Ota, Takeshi Seki*, Nobuhiro Nomrra**, Kazuhitu Suglmura", NoLuAisa Mizuki", Hirofuml Rkushima, Kimiyoshl Tsuji** and Hidotashi Inoko" Department of Legal Medicine, Second Department of Internal Medicine, Shinshu University School of Medicine, Nagano, "Department of Transplantation, Tokai University School of Medicine, Kanagawa, Japan
Key words: polymerase chain reaction - restriction fragment length polymorphism - sequencespecific oligonucleotide - DM1 and DPBl alleles - genotype Received 14 November 1990, revised, accepted for publication 28 May 1991
and suitable conditions for hybridization determined, which is technically demanding. The PCRRFLP method is practical and conventional for genotyping, but small fragments or bands located close to each other on the polyacrylamide gels sometimes obstruct precise analysis and some heterozygotes cannot be discriminated from each other (18). In this study we searched for informative restriction enzymes which have a single recognition site in some alleles, but none in other alleles in the amplified segments of the HLA-DQA1 or -DPBl genes (19-26), making reading of the generated RFLP band patterns much simpler. The PCRamplified DNAs digested with these restriction endonucleases (ApaLI, HphI, BsaJI, FokI and MboII for DQA1, Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII for DPBl) were subjected to electrophoresis. DQAl and DPBl genotypes were determined just by checking whether the amplified DNAs are mainly digested or not. Heterozygotes could also be analyzed with
Modified PCR-RFLP for DP and DQ
DPB1*0301 n~~i*o4oi DPBl*0402
DPB~*O~O~ DPgl*OBOl DPBl*0801 DPBl*0901 DPB1:lOOl DPBl*llOl DPBlt1301 DPBltl8Ol DFBl*lSOI DPBltl401 DPBlt1501 DWl*lBOl DPBl.1701
Table 1. Correlation between cleavage patterns obtained by 9 restriction endonucleases and DPB1 alleles Restriction endonucleases ~~
OPE1 allele 0401 1801 = 0402' 0201 0202 0801 = 1601 0501 0301 0601 1101 0101 0901 = 1701 1001 1301 1901 1401 1501
~
______~
Group
Bsp12861
Fokl
Ddel
BSall
BssHll
Or131
Rsal
EcoNl
Avall
A
1 1 1 1 0 1
1 1 1 1 1 1
0
1 0 0 0 0
1 1 1 1
0
0 1 1 1 0 1
0 0
0
0
1 1 1 1 1 1 1 0 1
1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1
0
I 0
1 1 1 1 1 1 0 0 0 1
1 1 0
1
0 C D E F G H I J
K L M N
0 P
0 0
1 1
0 0 1 1
0 0 1 0 1 0 0 0 0 0 0
0 1 0 0
0 0 0 1 0
1
0 0 0 1 1 0 0 1
0
0 1 1 1 0
0 0 0
0
0
0
1 1
1
1 1 1 1 1 1
0 0
0 1 1 0 0 0 0 0 1 1
0: not cleaved, 1: cleaved. *: DPB1'1801 and 0402 can be discriminated by detecting RFLP bands digested with Rsal. A 147bp fragment is positive for DPB1'1801, a l77bp fragment positive for OPBl"0402 and heterozygote OPB1'1801/0402 has both fragments (147 and 177 bp fragments). ': DPB1'0801 and ,1601 can be discriminated by detecting RFLP bands after digestion with Fokl (116bp positive for 0801 and 59bp positive for 1601). I: DPB1'0901 and 1701 can be discriminated by detecting RFLP bands after digestion with Fokl (116bp positive for 0901 and 59bp positive for 1701) (see Tables 2, 4, 5 and 6).
61
Ota et al. Table 2. Fragments (bp) detected by digestion of the PCR-amplified DPBl genes by 9 different restriction endonucleases Restriction Endonucleases
DPB1 alleles
Bspl2861
Fokl
We1
BsJl
BssHll
Cfrl31
Rsal
EcoNl
Avall
0401 0402 0201 1801 0202 0801 0501 0301 0601 1101 0101 0901 1001 1301 1901 1401 1501 1601 1701
262.37 262,37 262,37 262,37 153,109,37 299 190,109 299 299 299 299 268,31 268,31 299 299 268,31 262,37 299 268,31
186,57,56 183,59,57 183,5937 183,5937 183,59,57 183,116 183,59,57 299 242,57 242,57 183,116 183,116 183,116 183,116 183,116 299 242,57 183,59,57 183,59,57
299 299 299 299 166,133 299 166,133 299 299 299 299 299 299 299 166,133 299 299 299 299
246.32,21 246.32,21 246,32,21 246,3221 246,32,21 246,32,21 246.32.21 278,21 278,21 278,21 246,32,21 278,21 278.21 278,21 246.32,21 278,21 246.32.21 246.32.21 278.21
188,111 299 299 299 299 299 299 299 299 181,111 188,111 299 299 188.1 11 299 299 188,111 299 299
258,40 258,40 258,40 258,40 258.40 299 299 194.1 05 194,105 194.1 05 299 299 299 299 299 194,105 194,64,40 299 299
177.1 22 177,122 177,122 147,122,30 177.1 22 177,122 177,122 269,30 269,30 147,122.30 122,78,69,30 299 177,122 122,78,69,30 177,122 299 122,78,69,30 177,122 299
269,30 269,30 174,95,30 299 174,95,30 174,95,30 269,30 201,98 201,95,3 201,98 299 204,95 204,95 201,98 174,95,30 201,98 201,98 174,95,30 204,95
299 299 299 299 299 299 299 194,105 194.1 05 194,105 299 299 299 299 299 194,105 194,105 299 299
the method. This improved PCR-RFLP method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method, and so is a good alternative to the PCRSSO method.
Material and Methods DNA samples
70 DNA samples used in this study were distributed for Southern blot analysis in the Tenth International Histocompatibility Workshop. They were isolated from EBV-transformed HLA homozygous B-lymphoblastoid cell lines which were used in the previous study (10, 11). Genomic DNAs from 100 healthy Japanese volunteers were isolated by phenol extraction of sodium dodecyl sulfate (SDS)lysed and proteinase K-treated cells, as described earlier (27). PCR amplification
Genomic DNA (1 pg) was amplified by the PCR procedure with 2.5 units of the Taq DNA polymerase (Perkin Elmer Cetus Inc.) (28). The reaction mixture was subjected to 30 cycles of 1 min at 94"C, 1 min at 62"C, and 2 min at 72°C by automated PCR thermal sequencer (Iwaki Glass Inc.). The second exon of the DQAl gene was amplified by using the PCR primers, GH26 and GH27 at 1 pM and the second exon of the DPBl gene was amplified by using the PCR primers, DPB 10IN and DPB201 at 1 pM (Fig. 1).
62
Digestion with restriction endonucleases
After amplification, aliquots (7 pl) of the reaction mixture were digested with restriction endonucleases (ApaLI, HphI, FokI, MboII, Bsp 12861, DdeI, Cfrl31, RsaI, EcoNI, and AvaII; 5 units) at 37°C for 3 h after addition of appropriate incubac tion buffer. When digested with BsaJI or BssHII, the reaction mixture was incubated at 60°C or 50°C for 1 h, respectively. ApaLI, HphI, BsaJI, FokI, MboII and MnlI were used for digestion of the amplified DQAl gene and Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII
Table 3. Correlation between cleavage patterns obtained by 5 restrictionendonucleases and WA1 alleles Restriction endonucleases wA1 allele
0101 0102 0103 0201 0301 0501 0401 0601
Group
ApaLl
Hphl
A
1
0
0
0
0
B C D E F G
1 0 0 0 0 0
1 1 0 0 0 1
0 0 0 1 1 1
0 1 0 0 1 1
1
Bsall
Fokl
Mboll
0 1 0 0 0
0: not cleaved, 1: cleaved. DQA1 0101 and 0102 can be discriminated using Mnll. A 40bp fragment is positive for 0101 and negative for 0102. On the other hand, a 23bp fragment is positive for 0102 and negative for 0101 (see Table 11 and Fig. 3).
Modified PCR-RFLP for DP and DQ Table 4. Patterns of polymorphic fragments detected with Rsal enzyme for discrimination of heterozygotes between DPB1'1801 or 0402 allele and the other DPBl alleles Combinations DPBl alleles
040111801 040110402 180111801 180110402 040210402 020111801 020110402 020211801 020210402 0801 =160111801' 0801 = 1601/0402' 050111801 050110402 030111801 030110402 060111801 060110402 110111801 110110402 010111801 010110402 0901 = 170111801' 0901 = 170110402" 100111801 100110402 130111801 130110402 190111801 190110402 140111801 140110402 150111801 1501/0402
269 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
177 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 0 1 0 1 1 1 0 1 1 ' 1 0 1 0 1
147
122
1 0 1 1 0 1 0 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 1 0
1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0
1: positive, 0: negative. 5 RFLP bands digested with kkl can discriminate DPB1*0801/1801 (positive for 116 bp) and DPB1'160110201 (negative for 116bp). I: RFLP bands digested with Fokl can discriminate DPB1'080110402 (positive for 116 bp) and DPB1'160110402 (negative for 116 bp). *: RFLP bands digested with Fokl can discriminate DPB1'090111801 (positive for 116 bp) and DPB1'170110201 (negative for 116 bp). I: RFLP bands digested with Fokl can discriminate OPB1'0901/0402 (positive for 116 bp) and DP61'1701/0402 (negative for 116 bp).
were used for digestion of the amplified DPBl gene. Complete digestion of restriction enzymes was confirmed by including positive control DNAs with the HLA alleles which have cleavage sites for respective enzymes in the PCR-amplified regions. Acrylamide gel electrophoresis
Samples of the restriction enzyme-cleaved amplified DNAs were usually subjected to electrophoresis in 12% polyacrylamide gels in a horizontal minigel apparatus (Mupid, Cosmo Bio Co. Ltd.). When digested with MnlI enzyme, 15% poly-
Table 5. Patterns of polymorphic fragments detected with Fokl enzyme for discrimination of heterozygotes between DPB1'0801 or 1601 allele and the other DPBl alleles Combinations DPBl alleles
040110801 040111601 1801 =0402/0801' 1801 = 040211601H 020110801 601 0201/I 020210801 020211601 080110801 080111601 160111601 050110801 050111601 030110801 030111601 0601/a801 060111601 110110801 110111601 D10110801 010111601 090110801 170110801* 090111601' 170111601 100110801 100111601 130110801 130117601 190110801 190111601 140110801 140111601 150110801 ' 150111601
FokUbP)
183
116
59
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 0
0 1 1 1 1 1 1 1 0 1 1 1 1 0 1 0 1 0 1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1
1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 0
1: positive, 0: negative. *: RFLP bands digested with Rsal can discriminate DPB1'180110801 (positive for 147 bp) and DPB1*0402/0801 (negative for 147 bp). #: RFLP bands digested with Rsal can discriminate DPB1'18011 1601 (positive for 147bp) and DPB1'0402/1601 (negative for 147bp). ': 170110801 and 090111601 give the same RFLP band pattern and cannot be discriminated from each other.
acrylamide gel was used. Cleavage or no cleavage of amplified fragments was detected by staining with ethidium bromide.
Results Analysis of nucleotide sequences of PCR-amplified regions in the DPBl and DQA1 genes for allele-specific restriction
sites Fig. 1 summarizes the allelic variations in the 299bp PCR-amplified region of the second exon in the DPBl genes reported by published papers (21-26), where polymorphism in the DPB locus is 63
Ota et al.
primarily localized. These DNA sequences were used to search for restriction endonucleases which have a single cleavage site in some alleles (indicated by 1 in Table 1) but none in other alleles (indicated by 0 in Table 1) in the amplified region by computer analysis and 9 different restriction endonucleases, Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII, were selected for digestion' to detect allele-specificcleavage after PCR-amplification of the DPBl alleles (Table 1). Table 2 shows the sizes of the fragments detected by digestion of these restriction enzymes. Here, eight DPBl alleles (DPBl*0201, *0202,.*0401, *0402, *0501, *0801, Table 6. Patterns of polymorphic fragments detected with Fokl enzyme for discrimination of heterozygotes between DPB1'0901 or 1701 allele and the other OPBl alleles Combinations DPBl alleles 040110901 0401/1701 1801 = 0402/0901* 1801 = 0402/1 7Olx 020110901 020111701 020210901 020211701 080110901 160110901 080111701* 160111701 050110901 050111701 030110901 030111701 060110901 060111701 1 10110901 1101/1701 0101/0901 010111701 090110901 090111701 1701/1701 100110901 100111701 130110901 1301I1701 190110901 190111701 140110901 140111701 150110901 150111701
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 0
1 0
1 0 1 0 1 1 1 0 1 0 1 0 1 0 1 0 1 1 1 1 0 1 1 1 1 1 1 1 0 1 0
0 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 0 1 0 1 0 1 0 1 1 0
1 0 1 0 1 0 1 0 1
1: positive, 0: negative. *: RFLP bands digested with Rsal can discriminate DPB1'180110901 (positive for 147bp) and DP61*0402/0901 (negative for 147bp). #: RFLP bands digested with Rsal can discriminate DP61'180111701 (positive for 147bp. negative for 177bp) and DPB1'040211701 (negative for 147bp, positive for 177bp). *: 1601/0901 and 0801/1701 give the same RFLP band pattern and cannot be discriminated from each other as described in the foodnote of Table 5.
64
*1901 and *1601) have a cleavage site for EcoNI at the 30th nucleotide position from the 5' terminal and so, in the case of EcoNI, 269bp fragmentpositive alleles were also indicated by 0 for simplification in Table 1. For the same reason, 278bp BsaJI fragment-positive alleles were also indicated by 0 in Table 1. As shown in Table 1, these 9 different enzymes predict discrimination of 16 of 19 DPBl alleles, because the DPB1*1801 and DPB1*0402, DPB1*0801 and DPBl*1601, and DPB1*0901 and DPB 1* 1701 alleles give the same cleavage pattern. However, these three pairs of alleles can be discriminated by RFLP band patterns obtained with digestion of RsaI or FokI enzyme (Table 2, see also Tables 4 to 6). Digestion with RsaI restriction endonuclease gave a 147bp fragment for the DPB1*1801 and a 177bp fragment for the DPB1*0402 allele, and both fragments (147bp and 177bp) for DPBl* 1801/0402 heterozygote. Digestion with FokI restriction endonuclease gave a 116bp fragment for the DPB1*0801, a 59bp fragment for the DPB 1* 1601 allele and both fragments (1 16bp and 59bp) for DPB1*0801/1601 heterozygote. Further, digestion with FokI restriction endonuclease gave a 116bp fragment for the DPB1*0901, a 59bp fragment for the DPBl*1701 allele and both fragments (1 16bp and 59bp) for DPBl*O901/1701 heterozygote. In a similar way, the allelic variations in the 242bp PCR-amplified region of the DQAl genes were used to search for restriction endonucleases specific for each of eight DQAl alleles by computer analysis, and 5 different restriction endonucleases, ApaLI, HphI, BsaJI, FokI, and MboII, were selected to detect allele-specific cleavage after PCRamplification (Table 3). As shown in Table 3, these 5 restriction enzymes are expected to allow discrimination of all but two (seven out of eight) DQAl alleles because the DQA1*0101 and DQA1*0102 alleles give the same cleavage pattern. This problem was overcome by digestion with MnlI restriction endonuclease which distinguished the DQA1*0101 allele from the DQA1*0102 allele on the basis of the RFLP band patterns (see Fig. 3 and Table 11). A 40bp fragment is positive for the DQA1*0102 allele and negative for the DQAl*0101 allele. On the other hand, a 23bp fragment is positive for DQAl*O101 but negative for DQA1*0102. Digestion patterns by allele-specific restriction endonucleases in the amplified DPBl and DQAl genes from homozygous and heterozygous individuals
In order to test the method developed above, genomic DNAs from 70 HLA-homozygous B-cell lines with different DP and DQ specificities and from
Modified PCR-RFLP for DP and DQ Table 7. Cleavage patterns obtained by restriction enzymes in DPBl heterozygotes
Restriction Endonucleases
Combinations of DPBl alleles
Groups
0401I1801 =0402' 0401I0201 040110202 0401I0801 = 1601' 0401/0501 0401I0301 040110601 0401I1101 040110101 040110901= 1701 0401I1001 0401I1301 0401I1901 0401I1401 0401I1501 '1 801 = 0402/0201 '1 801 = 0402I0202 '1 801 = 0402/0801= 1601' '1801 = 0402I0501 7801 = 0402I0301 '1 801 = 0402I0601 '1 801 = 040211101 '1 801 =0402/0101 '1 801 = 0402/0901= 1701 '1 801 = 0402I1001 '1 801 = 0402I1301 '1 801 = 0402I1901 "1 801 = 0402I1401 '1 801 = 0402I1501 0201/0202 0201I0801 0201/0501 0201I0301 020110601 0201I1101 0201/0101 0201/0901= 170lX 0201I1001 0201I1 301 0201I1901 401 0201/I 0201I1501 0202/0801= 1601' 0202/0501 0202I0301 0202I0601 0202/1101 0202/010? .0202/0901= 1701 0202/1001 0202I1301 0202/1901 0202/1401 0202I1501 %80l= 1601/0501 WE01 = 1601I0301 %SO1 =160110601 '0801 = 1601I1101 %a01 = 1601I0101 '0801 =160110901=1701I
A/B A/C A/D A/E"
NF A/G A/n2) All A/J A/K AIL AIM A/N3) NO
A/P BIG B/O
BIE BIF BIG BIH B/l2' B/J BIK BIL B/M BIN BIO BIP
c/o
CIE CIF CIG CIH CII CIJ" C/K CIL CIM CIN"
c/o CIP
D/P' DIF DIG OIH D/I 01~3)
DIK D/L
DIM DIN
DIO DIP UF UG UH
UI UJ
OK
Bsp12861
Fokl
Ddel
BSdl
BssHll
Cfrl31
Ral
EWNl
Avail
1 1 1 2 1 2 2 2 2 1
-1 1 1
0 0 2 0 2 0 0 0
1
2 2 2 2 2 2 2 1 1 2 2 1 2 2 1 0 0 0 0 0 0 2 2 0 0 2 0 0 2 0 0 0 0 0 2 2 0 0 2
1
1
1 1 2 2 1 1 1 2 2 2 2 2 1 1 1 1 2 2 1 1 1 2 2 2 2 2 1 1 1 2 2 1 1 1 2 2 2 2 2 1 1 2 2 1 1 1 2 2 2 2 2 1 1 0 2 2 2 0 0
1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 2
0 2 2
0 0
1
2 2 1 1 1 1 2 1 2 2 2 2 1 1 2 2 1 1 1 2 1 2 2 2 2 1 1 2 2 1 1 2 1 2 2 2 2 1 1 2 2 1 1 2 0 0 0 0 2
1 1 2 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 2 1 1 2 1 1 1 1
0 0 0 0 2 0 0 0 2 0 2 0 0 0
0 0 0 0 2 0 0 2 0 2 0 0 0
0 0 0 0 2 0 0 2 1 2 2 2 2 2 2 2 1 2 2 2 0 0 0
0 0
1 1 1 1 2 2 2 1 2 2 2 1 2 1 1 1 1 1 2 2 2 1 2 2 2 1 2 1 1 1 1 2 2 2 1 2 2 2 1 2 1 1 1 2 2 2 1 2 2 2 1 2 1 1 2 2 2 1 2
0
0 2 0 0 0 0 2 2 0 0 2 0 0 2 0 0 0 2 2 0
2 0 2 2 2 0 2 2 2 2 2 2 2 2 2 0 2 2 2 0 2 2 2 2 2 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 2 1 1 1 2 1
0 0 0 2 2 2 0 0 0 0 0 2 2 0 0 0 0 2 2 2 0 0 0 0 0 2
2 0 0 0 2 2 2 0 0 0
0 0 2 2 0 0 2 2
2 0 0 0 0 0 2 2 0 2 2 2 0 0 contd.
Ota et al. Table 7 (contd.) Combinations of
Restriction Endonucleases
DPBl alleles
Groups
'0801 = 1601I1001 s0801= 1601/1301 $0801= 1601I1901 '0801 = 1601I1 401 $0801= 1601I1 501 050110301 0501/0601 0501I1 101 0501I0101 050110901 = 1701# 0501I1001 0501/1301 0501I1901 0501/1401 0501I1501 0301I0601 0301/1101 0301/0101 0301/0901=1701# 0301/1001 0301I1 301 0301/1901 0301I1 401 0301I1501 060111101 0601I0101 0601I0901 0601/1001 0601I1 301 0601I1901 0601I1401 0601I1 501 1101/0101 1101/0901=17Olx 1 101I1001 110111301 1 101I1901 1101/1401 1101/1501 0101/0901=1701# 0101I1 001 010111301 01 01I1 901 010111401 0101Il501 f0901= 1701I1 001 x0901= 1701I1301 %901= 1701I1 901 #0901= 1701I1 401 80901=1701/1501 1001I1301 1001I1 901 1001I1 401 1001I1 501 1301/1901 1301I1 401 1301I1501 1 901I1401 1901I1501 1401I1501
EIL EIM EIN
fY0 UP FIG FIH FII FIJ FIK FIL FIM F/N
fikl
Ddd
BMl
BSSHll
Or131
Rsal
2 0 0 2 2 2 2 2 2 1
1 1 1 2 1 2 1 1 1 1 1 1 1 2 1 2
0 0 2 0 0 2 2 2 2 2
2 2
0 2 0 0 2 0 0 2 2 0 0 2 0 0 2 0 2 2 0 0 2 0 0 2 2 2 0 0 2 0 0 2 1 2 2 1 2 2 1 2 2 1 2 2 1 0 2 0 0
0 0 0 2 2 2 2 2 0 0 0 0 0 2 2 1 1 2 2 2 2 2 1 1 1 2 2 2 2 2 1 1 2 2 2 2 2 1 1 0 0 0 0 2 2 0 0 0 2 2 0 0 2 2 0 2 2 2 2 1
1 1 1 2 1 1 1 1 1 2 1
1
2 2 1 1 0 0
F/O
F/P GIH G/I GIJ GIK GIL GIM GIN GI0
G/P H/I HIJ HIK HIL HIM HIN HI0
H/P IIJ IIK IIL IIM IIN 110
IIP JIK JIL JIM JIN JIO JIP WL WM WN WO UM UN
uo
2
2 2 0 0 2 2 0 0 2 2 0 0 2 2 0 2 2 0 0 2 2 2 2 0 0 2 2 1 2 2 1
2
0
2
0 0
1 1 0 2 2
UP MIN MI0
MIP
2
NIO NIP
2 1
OIP
2
2 2 1 2 2 0 0 0
0
1 2 2
WP
~-
Bsp12861
2 2 0 2 1 1 1 1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 2 1 1 1 2 1 1 2 1 2 1 2
2
0 0 0 0 0 0 0 2 0 0
0 0 0 0 2 0 0 0 0 0 2 0 0 0 0 2 0 0 0 2 0 0 2 0 0 2 2
0
1 2 1 2 2 2 1 2 2 2 1 2 1 0 0 2 0 0 0 2 0 2 0 2 0 0 0 2 0 2 2 0 0 0 2 0 2
2 2 2 1 2 1
0
0 2
0 2
2
0 2 0
2
2 2 0 2 2 1 2
0 0 2
2 2 1 0 2 2
1
1 2 1 1 1 1 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 2 1 1 1 2 1 2 1 1 1 2 1 2 2
2 0 2 1
1
2 1 1 2 1
2 1 2
EcoNl
Avafl
1
0 0 0 2 2 2
1 1 1 1 2 2 2 0 2 2 2 2 2 2 1 1 2 1
1 1 1 1 1 1 2 1 1 1 1 1 1
2 1 1 1 1 1
1 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 2 0 0 0 0 0 2 2 1 1 2 2 2 2 2 1 1 1 2 2 2 2 2 1 1
2 2 2 2 2 1 1 0 0 0 0 2 2 ' 0
0 0 2 2 0 0 2 2 0 2 2 2 2 1
~
0: not cleaved, 1: cleaved, 2: both for heterozygote. Following four combinations"-" give the same cleaving RFLP patterns obtained with 9 restriction endonucleases but can be distinguished by reexamination of RFLP band patterns as shown in Table 8. AUCJ'' can be discriminated by RFLP band patterns obtained by Fokl and Rsall. AWBI') can be discriminated by RFLP band patterns obtained by Rsal. ANIDJ3) can be discriminated by RRP band patterns obtained by Bspl2861. CNIDE') can be discriminated by RFLP band patterns obtained by Bspl2861 and Fokl. *: DP61'1801 and 0402 can be discriminated by detecting RFLP bands obtained by Rsal (Table 4). I: DPB1*0801 and 1601 can be discriminated by detecting RFLP bands obtained by Fokl (Table 5). #: OP61*0901 and 7701 can be discriminated by detecting RFLP bands obtained by Fokl (Table 6). Only one combination of 0801I1 701 and 0901I1 601 cannot be discriminated by any restriction enzymes so far examined.
66
Modified PCR-RFLP for DP and DQ Table 8. RFLP patterns detected with Fokl, Rsal and Bsp12861 restriction endonucleases Combinations of DPBl alleles
groups
0401/0801 0401/1601 0201/0101
AE AE
CJ
Fokl 183
116
122
78
1 1 1
1 0 1
1 1 1
0 0
Combinations of DPBl alleles
groups
0401/0601 1801/1101 0402/1101
AH El 81
groups
0401/1901 0202/0101
AN
269
177
122
1 0 0
1 0 1
1 1 1
Bspl2861 262
153
1 0
0 1
DJ
Combinations of DPBl alleles
groups
0202/0801 0202/1601 0201/1901
DE DE CN
1
Rsal
Combinations of DPBl alleles
Rsal
Bspl2861
Fokl
262
153
116
59
0 0 1
1 1 0
1 0
1 1
0: negative, 1: positive.
100 healthy Japanese individuals were subjected to amplification of the DQAl and DPBl genes, followed by digestion with the restriction endonucleases described and electrophoresis in 12% polyacrylamide gels. HLA-DPB1
16 of 19 DPBl alleles were found to be discriminated on the basis of the cleavage patterns with 9 different endonucleases using 70 HLA homozygous B-cell lines as expected from Table 1, although no B-cell lines with the 1101, 1401, 1801 or 1901 allele are available. Three pairs of indistinguishable alleles DPBl*1801 and DPBl*0402, DPB1*0801 and 1601 and DPB1*0901 and 1701 can be discriminated by RFLP bands detected with RsaI or FokI restriction endonuclease no matter what allele is combined with these alleles, as mentioned above (Tables 4,5 and 6). 16 standard DPBl PCR-RFLP patterns give 120 heterozygous combinations (Table 7). Note that heterozygotes carry both cleaved and uncleaved amplified bands, indicated by 2 here. Four pairs out of these combinations cannot be distinguished by cleavage patterns with 9 restriction enzymes (DPB1*0401/
Figure 2. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DPBl genes obtained from DNAs of 4 normal individuals after digestion with 9 restriction enzymes. Determination of their genotypes was based on Tables 1,4,5,6,7 and 8. The size of the amplified region of the DPBl exon 2 is 299 bp as indicated. Two EcoNI-digested samples, DPB1*0501/0501 and 0402/0501 gave a little bit lower band (269bp), but were indicated by 0 for the reason described in the text.
0801 = 1601 and 0201/0101,0401/0601 and 1801= 0402/ 1101,0401/ 1901 and 0202/0101,0202/0801= 1601 and 0201/1901). However, these four pairs can be discriminated by reexamination of RFLP band patterns obtained after treatment of FokI, RsaI or Bsp1286I enzyme (Table 8). DPBl*0401/ 0801, 0401/1601 and 0201/0101 heterozygous alleles can be discriminated by the presence of the 116bp fragment digested with FokI enzyme and the 78bp fragment digested with RsaI enzyme. Discrimination of heterozygous DPB 1*0401/0601, 1801/1101 and 0402/1101 genotypes can be done by examining the presence of the 269bp or 177bp fragment digested with RsaI enzyme. DPB1*0401/ 1901 and DPB1*0202/0101 can be distinguished by examining the presence of the 262bp or 153bp fragment after digestion with Bsp1286I enzyme. Lastly, heterozygotes between DPBl*O201/0801, 0202/1601 and 0201/1901 can be discriminated by examining the 262bp or 153bp fragment with Bsp1286I and the 116bp fragment with FokI enzyme. Heterozygotes between DPBl * I80 1 or *0402 and the other DPBl alleles can be discrimi67
Ota et al. Table 9. HLA-OPE1 and DQAl allele frequencies in Japanese DPBl
n
Freq(%)
G.F.(%)
WA1
n
0101 0201 0202 0301 0401 0502 0501 0601 0801 0901 1001 1101 1301 1401 1501 1601 1701 1801 1901
0 32 9 7 9 23 57 1 0 19 0 0 3 2 0 1 0 0 0
0 32 9 7 9 23 57 1 0 19 0 0 3 2 0 1 0 0 0
0.0 17.5 4.6 3.6 4.6 12.3 34.4 0.5 0.0 10.0 0.0 0.0 1.5 1.o 0.0 0.5 0.0 0.0 0.0
0101 0102 0103 0201 0301 0401 0501 0601
22 29 40 1 76 1 14 3
~~
Table 10. Cleavage patterns obtained by restriction enzymes in W 1 heterozygotes
heq(%) G.F.(%) 22 29 40 1 76 1 14 3
11.7 15.7 22.5 0.5 51.O 0.5 7.3 1.5
~
Total number examined is 100 G.F.: Gene Frequency, G.F was estimated according to the Beinstein formula.
G.F.=l-(l-freqE
nated by detecting RFLP bands after digestion of RsaI enzyme (Table 4) and heterozygotes between DPB1*0801 or *1601, or DPB1*0901 or *I701 and the other DPBl alleles can be discriminated by checking RFLP fragments detected with FokI enzyme (Tables 5 and 6). In this way, all of 189 homozygous and heterozygous combinations except for DPBl*1601/0901 and DPB1*0801/1701 can be clearly defined. DPBl*1601/0901 and DPB1*0801/1701 give the same RFLP pattern and cannot be discriminated from each other by any restriction enzymes so far available. In order to assess the possibility of DPBl genotyping on the basis of the cleavage patterns predicted by the combination of two standard patterns of DPBl homozygotes as shown in Table 1, genomic DNAs from 100 normal, randomly selected Japanese individuals were analyzed (Fig. 2). RFLP patterns obtained with 9 restriction endonucleases were matched with one of the patterns given in Tables 1, 4, 5, 6, 7 and 8, and all of their DPBl genotypes could be distinctively defined, and their gene frequencies are shown in Table 9. HLA-DQA1
Seven of eight DQAl alleles were found to be discriminated on the basis of the cleavage patterns with 5 restriction enzymes using 70 HLA homozygous B-cell lines as expected from Table 3. In this improved PCR-RFLP method, seven DQA PCR-RFLP patterns (Table 3) can be combined in 68
Combinations of
Restriction endonucleases
DQA1alleles
Groups
0101 = 0102/01OY 0101 = 0102/0201' 0101 = 010U0301' 0101 = 0102/0501* 0101 = 010210401' 0101 = 0102/0601' 0103/0201 0103/0301 0103/0501 0103/0401 0103/0601 0201/0301 020110501 0201/0401 020110601 0301I0501 0301I0401 0301I0601 0501/0401 0501I0601 0401/0601
A/B AJC AID
AJE AIF
AJG B/C BID BIE B/F BIG C/D
CIE CIF C/G
D/E OIF DIG
UF WG FIG
ApaLl
Hphl
Bsall
Fokl
Mboll
1 2 2 2 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 0 0
2 2 0 0 0 2 1 2 2 2 1 2 2 2 1 0 0 2 0 2 2
0 0 0 2 2 2 0 0 2 2 2 0 2 2 2 2 2 2 1 1 1
0 2 0 0 2 2 2 0 0 2 2 2 2 1 1 0 2 2 2 2 1
2 0 2 0 0 0 2 1 2 2 2 2 0 0 0 2 2 2 0 0 0
0: not cleaved, 1: cleaved, 2 both for hetrozygote. ': DQA1 0101 and 0102 can be discriminated by using Mnll enzyme (see Table 11).
28 homozygous and heterozygous ways (Table 10). All the 28 combinations can be distinguished from each other. If Mnll restriction endonuclease is included, the DQA 1*O 101 and DQA 1*O 102 alleles can also be distinguished (Table 11, Fig. 3) and all
Table 11. Patterns of polymorphic fragments detected with Mnll enzyme for discrimination of homozygotes or heterozygotes including MA1'01 01 or 0102 alleles Combinations of
Mnll(bp)
wA1 alleles 0101 0101 0102 0103 0201 0301 0501 0401 0601 0: negative, 1: positive.
0101 0102 0102 0101 0102 0101 0102 0101 0192 0101 0102 0101 0102 0101 0102
23
40
1 1 0 1 0 1 1 1 1 1 0 1 0 1 0
0 1 1 0 1 0 1 0 1 1 1 1 1 1 1
Modified PCR-RFLP for DP and DQ
of 36 homozygous and heterozygous combinations can be unequivocally discriminated. 20 cell lines belonging to A group (DQA1*0101 or DQA1*0102) in our previous study (10) could be further characterized by using MnlI and classified into DQAl *O 101/O101, DQA 1*0101/0102, or DQA1*0102/0102 genotype (Fig. 3, DQA1*0101/ 0102 is not shown). Their genotypes were found to correlate well with the Dw specificity. Namely, the DQA1*0101 allele is associated with the Dwl, Dw9 or DB2 specificity and DQAl*0102 allele is associated with the Dw2, Dw21 or Dw19 specificity. Genomic DNAs from 100 healthy Japanese individuals are amplified and typed for DQAl genotype by this modified PCR-RFLP method (Fig. 4). All of their DQAl genotypes and gene frequencies could be determined unequivocally (Table 9). Discussion
The modified PCR-RFLP method presented here which depends mainly on cleavage or no cleavage of the amplified segment by informative restriction enzymes provides a simpler and more practical
Figure 3. Patterns of polymorphic restriction fragments obtained with digestion of the PCR-amplified DQA1*0101 and 0102 genes by MnlI enzyme. DNA samples were electrophoresed in a 15% polyacrylamide gel.
technique than our previous PCR-RFLP method and can identify HLA alleles of individuals homozygous or heterozygous at the DQAl and DPBl loci. Flow charts for DQAl and DPBl genotyping by this method are illustrated in Fig. 5. All the possible 36 and 189 combinations of DQAl and DPB 1, respectively, including homozygotes and heterozygotes, can be unequivocally discriminated with the restriction endonucleases selected here with only one exception, DPB1*1601/0901 and *0801/1701 which cannot be distinguished even by the PCR-SSO technique. This indistinguishable combination can be discriminated only by using allele-specific primers for the DPB 1* 1601 or *0801 allele. All of the restriction enzymes used here are commercially available, but some of them are expensive at present (e.g. AvaII and MnlI). Such enzymes can be replaced by other cheaper restriction enzymes as described in the previous paper (10, 11) or omitted depending on the typing sensitivity to be required. One major problem of the PCR-RFLP technique is incomplete or partial digestion of the PCR products by restriction enzymes, which will obscure definite assignment of HLA alleles. As included in any other typing methods, this problem can be
Figure 4 . Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DQAl genes obtained from DNAs of 6 normal individuals after digestion with 6 restriction enzymes. Determination of their genotypes was based on Tables 3, 10 and 11. The size of the amplified region of the DQAl exon 2 is 242 bp as indicated. DQA1*0201, 0401, 0501 and 0601 alleles have a 3bp deletion in the PCR-amplified exon 2 region and so some heterozygotes (e.g. DQAl*OlO2/0601) give two PCR amplified bands with different sizes detected when there are no cleavage sites of either allele for the restriction enzymes used (e.g. B: MboII).
69
Ota et al.
0:z Tables 1.4.5.6
It not
\
/
It not
\
/
DPBl G s n o t Y p e
Figure 5 . Flow chart of the modified PCR-RFLP method for determining genotypes of DPBl and DQAl. X: arbitrary allele.
overcome just by preparation of positive control DNAs for ensuring that the digestion is complete. Further, PCR primers incorporating recognition sites for restriction enzymes used here will be more useful as internal controls. The DQAl and DPBl alleles of 70 HLA-homozygous B-cell lines used in the Tenth International Histocompatibility Workshop defined by this PCR-RFLP method were found to be in complete agreement with those defined by the PCR-SSO method (N. Mizuki et al., unpublished, A. Kimura, unpublished). Thus, the PCR-RFLP method provides a useful, reproducible and accurate genotyping technique for HLA alleles. These genotyping analyses can be easily managed with a personal computer program. This modified PCR-RFLP method eliminating the need for radioactive or non-radioactive labeling of multiple SSO probes is a good alternative to the PCR-SSO method and can be substituted for the conventional serological and cellular techniques. This modified PCR-RFLP technique has also been successfully extended and applied to DQBl and DRBl genotyping for HLA homozygous or heterozygous individuals in larger trials (N. Nomura et al., in press, M. Ota et al., submitted). Acknowledgments
We thank Dr. J. Trowsdale of Imperial Cancer Research Fund for critical reading of the manuscript and Dr. J. H. Lee of One Lamda for giving important information of restriction enzymes. This work was supported by a grant-in-aid for scientific research in 1990 from the Japanese Ministry of Education. References 1. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA.
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