HLA-DRB1 genotyping by modfied PCRRFLP method combined with group-specific pruners M. Ota, T. Seki, H. Fukushima, K. Tsuji, Inoko. HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers. Tissue Antigens 1992: 39: 187-202. , Abstract: We previously introduced HLA-DQAl ,-DPB1 and DQBl genotyping with the modified 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. In this study, 43 HLA-DRB 1 aneles, excluding DRB 1* 1103 and * 1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB1*0701 and DRB1*0702 as they are identical for the second exon of DRBI. For DRIDRBI, DR2-DRB1, DRCDRBl, DR7-DR1, DR9-DRB1, DRwlODRBl or DRw52 associated antigens (DR3, wll, w12, w13, w14, and DRw8)-DRBl gene amplification, the second exon of the DRBl gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and 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, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB 1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCRRFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.
Introduction The HLA class I1 genes (HLA-DR, -DQ, -DP), located in the short arm of chromosome 6, specify heterodimeric glycoproteins involved in the regulation of the immune response (1). These highly polymorphic molecules bind to foreign or self antigenic peptides and present them to antigen-specific T cells in a self-restricted fashion (2-5). Accurate identification of their polymorphisms is a prerequisite for determination of the functional role of HLA in immunoreactivity (6) and transplantation immunity (7-8), as well as in the susceptibility to autoimmune diseases (9). In the DRB region, five DRB (DRB1, DRB2, DRB3, DRB4 and DRB5)
Masao Ota', Takeshi Seki', Hirofumi Fukushima', Kimiyoshi Tsuji' and
Htdstoshi Inoko' 'Department of Legal Medicine, *Second Department of Internal Medicine, Shinshu University School of Medicine, Nagano. and 'Department of Transplantation, Tokai University school of Medicine, Kanagawa, Japan
Key words: PCR: polymerase chain reaction RFLP: restriction fragment length polymorphism - SSO: sequencespecificoligonucleotide - DRBl alleles - genotyping Received 16 September, revised, accepted for publication 4 December 1991
loci have been identified and these DRB genes, except for a pseudogene, DRB2, are known to encode specific DR chain molecules. Among them, the DRBl genes is thought to be the most important in controlling the immune response as well as the main alloantigen determinants. There are now as many as 46 DRBl alleles known,among which 43 have different amino acid sequences in the /3 1 domain-exon (10). DR specificities are usually determined by serological procedures using alloantisera or monoclonal antibodies. The availability of these antibodies is limited in specificity, quantity and sensitivity for full DRB typing (1 1). Furthermore, the cellular assay procedure (12) used for definition of Dw specificity is time-consuming and 187
Ota et al. Table 1. PCR primers for allele- or groupspecific amplification of the DRBl gene ~
Gene DRBlS primer
DRBl 3’ primer
Primers for DR2 for DR4 for DR9 for DRl forDR7 for DR10 DR3
5’R2 5‘R4 539-1 5’Rl SR7 SR10
for DR6 DR5 DR8
5’R3568
3’R
codon matched
Sequences (5’ to 3’)
PCR-products
7-1 3 6-1 3 8-1 5 25-32 25-32 25-32
TTCCTGTGGCAGCCTAAGAGG GmCTTGGAGCAGGllAAAC GPAGCAGGATAAGmGAGTG GGTTGCTGGAAAGAECATCT AGTTCCTGGAAAGACTCTTCT GGTGCTGGAAAGACGCGTCC
(261 bp) (263bP) (256bP) (206bp) (206bp) (206bP)
5-1 2
ACGmCTTGGAGTAClCTACG
(265hp)
87-93
complicated by the limited availability of the typing reagents, homozygouS typing cells (HTCs). Restriction fragment length polymorphism (RFLP) analysis with a genomic Southern hybridization technique allows the identification of DRB alleles at the gene level, but requires a large amounts of high-molecular weight DNAs and a complicated procedure; also, the interpretation of RFLP band patterns is not straightforward. The limitation of these methods for identification of DRB polymorphisms has been overcome by the advent of polymerase chain reaction (PCR) technique
CCGCTGCACTGTGAAGCTCT
(13-17). The PCR method permits precise and direct analysis of allelic variations with as little as lng of genomic DNA. The PCR-RFLP method we reported previously (18-20) 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 the majority of heterozygotes cannot be discriminated from each other (21). These problems have been overcome by the modified PCR-RFLP method of incorporating informative restriction enzymes which have a single recognition site in some alleles, but none in other alleles in the amplified segments for the DPB1, DQAl and DQB1 genes (22-23) and thus reading of RFLP band patterns generated has become much simpler and easier. In this study, this modified PCR-RFLP method was applied to DRBl genotyping along with 7 pairs of group-specific primers. The PCRamplified DNAs digested with restriction endonucleases (AvaII and PstI for DR1 group; FokI, Cfrl3I and HphI for DR2 group; SacII, AvaII, HinfI, HaeII, HphI and MnlI for DR4 group; AvaII, FokI, KpnI, HaeII, Cfrl31, SfaNI, SacII, BsaJI, ApaI, HphI and RsaI for DR3,5,6 and 8 group) were subjected to electrophoresis. DRBl Table 2. Correlation between cleavage patterns obtained by two restriction endonucleases and DRB1 homozygous and heterozygous alleles for OR1 Combinations of
Figure 1. The PCR products were amplified by each pair of the group-specific S’primers (SR1, S’R10, SR2, S’R4, S’R7, SR9 and S‘R3.568) and common 3’(3’R) primer. DNAs were extracted from HTCs and a healthy donor cells having DRwlOIDR2 heterozygote as indicated.
188
Restriction endonucleases
DRBl alleles
Groups
Avall
Pstl
0101/0101 0102/01 02 0103/0103 010110102 010110103 0102/0103
NA
1 1 0
1 0
BIB
c/c
A/B NC BiC
1
1 2
2 2
1 2
0: not cleaved, 1: cleaved, 2: both for heterozygote.
Modified PCR-RFLP for DRBl International Histocompatibility Workshop. They were isolated from EBV-transformed HLA homozygous B lymphoblastoid cell lines which were the same ones used in the previous study (23). Genomic DNAs from healthy Japanese volunteers were isolated by phenol extraction of sodium dodecyl sulfate (SDS)-lysed and proteinase K-treated cells, as described earlier (24). As no homozygous DRwlO cell line was available, a DRwlO heterozygote (DR2/DRwlO) was included for obtaining the standard RFLP band pattern of DRwlO.
Table 3. Cleavage and RFLP patterns obtained by three restriction endonucleases in combinations of DRBl homozygous and heterozygous alleles for OR2 specificity
-~ ~
Combinations of
Restriction endonucleases Hphl (bp)
ORB1 alleles
Groups
Fokl
Cfrl31
120
109
150111501 1502/1502 1601I1601 1602/1602 1 5 0 1502 ~ 1501I1601 1501I1602 1502/1601 1502/1602 1601/1602
NA BIB C/C
1 1 0 0 1 2 2 2 2 0
1 1 0 1 1 2 1 2 1 2
+
+ +
~~
DID
A/B NC NO 6IC BID C/D
+
+ +
-
-
+ + + + +
PCR amplification
Genomic DNA (200-300 ng) was amplified by the PCR procedure with 2.5 units of the Taq DNA polymerase (Perkin Elmer Cetus Inc.) (25). The reaction mixture (genomic DNA, PCR buffer; 50 mM KCl, 2.5 mM MgCl,, 10 mM Tris-HC1 pH8.4, 0.01% gelatin, 0.02% N(Nonidet)P-40; 200 pM each of dATP, dCTP, TTP and dGTP; 1 pM of each of the primers) and distilled water for a total volume 100 pI in a 1.5 ml eppendorf tube (Eppendorf Co.) was covered with 50 pl of mineral oil to prevent evaporation and was subjected to 30 cycles of 1 min at 94'C, I min at 6OoC, and 2 min at 72°C by automated PCR thermal sequencer (Iwaki Glass Inc.). For DR1 and DR9 amplification, annealing was performed at 55°C. The second exon of the DRBl gene was amplified using 1 of 7 groupspecific 5' primers (5'R2,5'R4,5'R9- I , 5'R 1, YR7, SR10 and YR3568) along with the common 3' primer (3'R) as shown Table 1.
+
+
~~
0: not cleaved, 1: cleaved, 2: both for heterozygote. +: positive, -: negative.
genotypes were determined mainly by checking whether the amplified DNAs are digested or not. Heterozygotes could also be analyzed with the method. This modified PCR-RFLP method resolves the multiple subtypes within the major DR groups and is technically simpler, cheaper and more practical for routine HLA typing work than our previous PCR-RFLP method, and thus is a good alternative and a complementary technique to the PCR-SSO method.
Material and Methods DNA samples
Seventy DNA samples used in this study were distributed for Southern blot analysis in the Tenth
Table 4. Cleavage and RFLP patterns obtained by six restriction endonucleases in DR4-DR61 hornozygous alleles Restriction endonudeases
Group
Hphl (bp) DR4-DRB1 alleles Sacll Avall Hinfl Haell 120 109
B
0
-
+
+
-
O
+
-
-
+
1 1 1
0 0 0
0
1
0
0
0
0403 0407
0
1 1
0 0
0 0
+
0
0405 0409 0410
1 1 1
1 1 1
0
1 1 1
-
+
0 0
-
+
+
-
+
-
-
+
0406
0
1
1
0
0
1
0
1
0402 ~
1 1
1 0
DRBM3101/0101 oRBrolo2/0102
HOM2
M2070782
Figure 2. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRI-DRBl genes obtained from DNAs of two HTCs after digestion with AvaII and PstI enzymes. Determination of their genotypes was based on Table 2. The size of the amplified region of the DRI-DRBI exon 2 is 206 bp as indicated.
C ~~
-
+
E F ~
0411 ~~~
-
+
- +
~~~
0
113 107
1 1 1
0401 0404 0408
A
Mnll (bp)
- +
~~
0:not cleaved, 1: cleaved, +: positive, -: negative.
189
Ota et al.
Digestion with restriction endonucleases After amplification, aliquots (7 pl} of the reaction mixtures were digested with restriction endonucleases (AvaII and PstI for DRl-DRB1; FokI, Cfrl3I and HphI for DR2-DRB1; SacII, AvaII, HinfI, HaeII, HphI and Mn1I for DRCDRBl; AvaII, FokI, KpnI, HaeII, Cfrl31, SfaNI, SacII, BsaJI, ApaI, HphI and RsaI for DR3,5,6 and 8DRB1 and 1-2 units) for 1 to 3 h after addtition of appropriate incubation buffer at suitable temperature. Complete digestion of restriction enzymes was confirmed by inluding positive control DNAs with the HLA alleles which have cleavage sites for respective enzymes in the PCR-amplified regions.
Table 5. Cleavage patterns obtained by restriction enzymes in combinations of DR4DRBI heterozygous alleles Combinations of
0403 (C) 0407 0401 0404 0408
0402 (B)
0403 (C)’ 0407
1 1 1 1 I
1
0 0
Fokl
1 1 1 1 1 1
Restriction endonodeases
DRB1 alleles Sacll
Avall
Hinfl
Haell
2
1
0
0
0405 0409 (0) 0410 0406 (E)
1
1
0
2
2
1
2
0
0411 (F)#
2
1
0
2
0403 (C) 0407
2
2
0
0
0
2
0405 0409 to) 0410 0406 (E)
1
2
2
2
2
0
0411 (F)
2
2
0
2
0405 0409 (D)# 0410
2
1
0
2
0406 (E)
0
1
2
0
0411 (F)
0
1
0
2
0405 0409 (0)’ 0410
0406 0411 (F)
2 2
1
2
2
1
0
1
0406 (E)
0471 (F)
0
1
2
2
0:not cleaved, 1: cleaved, 2: both for heterozygote. (Cr 0403,0407and (Dy 0405,0409,0410can be (A)’ 0401,0404,0408, distinguished by the use of Hphl and Mnll restriction enzymes (Table 6), or Sacll+Haell, Sacll+Hphl, Haell+Hphl or Haell+Mnl I double-digestion technique (Table 7). Combinations (#) of AF (0401,0404 or 0408/0411)to CD (0403 or 0407/0405.0409 or 0410) can be discriminated from each other by RFLP band patterns obtained after Hphl and.Mnll enzymes [Table 6), or Sacll+Hphl or Sacll+Haell double digestion (Table 7).
0 1
Cfrl31
Acrylarnide 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.). Cleavage or no cleavage of amplified fragments was detected by staining with ethidium bromide.
Hphl Figure 3. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRZ-DRBI genes obtained from DNAs of 8 HTCs after digestion with FokI, Cfrl3I and HphI enzymes. Determination of their genotypes was based on Table 3. The size of the amplified region of the DRZDRBI exon 2 is 261 bp as indicated.
190
Results Groupspecific amplification Fig. 1 shows the products of PCR amplification by 7 pairs of the group-specific 5’ primers and common 3’ primers (Table 1). Twelve genomic
Modified PCR-RFLP for DRBl Table 6. Patterns of polymorphic fragments detected with Hphl and Mnll enzymes for discrimination of heterozygotes between DRB1*0401, 0404 or 0408. DRB1’0403 or 0407, or DRB1.0405, 0409, or 0410 and other DACDRBl alleles
0401 0404 0401 0404 0408
Mnll (bp)
Hphl (bp)
Combinations of DRBl alleles
109
+ + + +
0401 0404 0408 0408
-+
0402
0401 0404 0408
0403
0401 0404 0408
0407
0401
0405 # 0409 0410 $
0404
0405 & 0409 S 0410
0408
0405 0409 # 0410 &
0401 0404 0408
0406
0401 0404 0408
+ +
-
+ + +
+ ~~
~~
+ +
120
107
113
-
-
+
+ + +
+ + +
-
+ ++ + +
+
+ -
+
~~
-
*
-
-
+
-
+ + + +
+
+ + -
-
+
+
+ + + + + + -
+ + +
+ + + +
+
+ + + +
+
! 0411 ? Y
+ -
+ +
+ +
0403
0405 Y 0409 ! 0410 ?
0407
0405 0409 0410 V
+ + + + + +
+ + + -
+ + + +
0402
0403 0407
+
+
+
+ + +
+ +
+
+
+
+
+
+ +
+
-
-
+ +
+
-
+ + + + -
-
-
+ +
0403
0407
+
.f
+ + +
0403 0407
0406
-
.t
+
0403 0407
0411
.f
+
+
+
+
+
-
0405 0409 0410
+
-
+
0402
+ -
+
+ + + +
+
-
-
+
+ -
Cont. table 6. Hphl (bp)
Combinations of DRB1 alleles 0405 0409
Mnll (bp)
109
120
107
113
0409 0410
+
-
+ +
+ -
0410
+
+
+
+
+
+
-
+
+
~~
+
0405 0409 0410
0406
0405 0409 0410
0411
~~~
+
+ + + + +
+ + -
~~~
~
+ + + + +
+ -
+ -
~~
-: negative, +: positive. Combinations of 0401/0411 and 0403/0409 (!), 0404/0411 and 0403/0410 (?), 0408/0411 and 0403/0405 or 0407/0410 (V) can be distinguished by RFLP bands obtained by doubledigestion with Sacll+Haell. Combinations of 0403/0405 and 0407/0410 (V) and 04031 0408 and 0404/0407 [‘) can be distinguished by doubledigestion with Sacll+Hphl. Combinations of 0401/0410 and 0404/0409 (S)and 0404/0405 and 0408/0410 (a) can be distinguished by doubledigestion with Haell+ Hphl. Combination of 0401/0405 and 0408/0409 (#) can be distinguished by doubledigestion with Haell+Mnll (See Table 7).
DNAs were extracted from HTCs distributed at the Tenth International HLA Workshop (HOM2,9005 on lane 1; WT8,9017 on lane 3; KASOII,9009 on lane 4; SAVC,9034 on lane 5; MOU,9050 on lane6; DXB,9075 on lane 7; VAVY,9023 on lane 8; BM21,9043 on lane 9; BM15,9040 on lane 10; OMW,9058 on lane 11 and TEM,9057 on lane 12) and healthy donor cells having DRwlO/ DR2 heterozygote on lane 2. These DRBl group-speficific primers (5’R2 to 5’R3568) were confirmed as amplifying the DRBl genes selectively from the DR2, DR4, DR9, DR1, DR7, DRw 10 and DRw52-associated antigens (DR3,
*
: Hphl
7 :S a d
Figure 4. The PCR-RFLP method can show linkage between two polymorphic sites. The nucleotide sequences are identical between DRB1*0403 and 0407 and between D R B l * W and 0408 except at the HphI(*) (in the right end) and Sac11( V ) sites. A characteristic feature of genetic polyrnorphisms displayed by the HLA genes is that their alleles can be distinguished by differing in combinations of variable regions by taking patchwork structure in the first domain exon. In the PCR-RFLP method double digestion (in this case, HphI+SacII) gives the information on the linkage between two polymorphic sites (here, the Sac11 and righ-end HphI sites) by the appearance of the cleaved fragment (in this case, a 47 bp fragment) which allows unequivocal discrimination between such heterozygotes.
191
Ota et al. DR5, DR6 and DR8), respectively. The DR7, DR9 and DRwlO alleles which have no suballele (DRB1*0701 and 0702 have the same nucleotide sequences in their j3l domain exons) can be typed simply by the presence of amplified bands generated with these group-specific primers (more exactly, allele-specific primers in these cases) as DRBl*O701 or 0702, 0901 and 1001, respectively. DR1-DRB1 genotyping
The DNA sequence allelic variations in the 206bp PCR-amplified region of the j3l domain exon in the D R l -DRB 1 genes (lo), where polymorphism in the DR 1-DRB 1 locus is primarily localized, were used to search for restriction' endonucleases which have a single cleavage site in some alleles (indicated Table 7. RFLP patterns detected with Sacll+Haell. Sacll+Hphl, Haell+Hphl or Haell+ Mnll doubledigestion for discrimination among 9 pairs of DR4 heterozygotes Combinations of
Sacll+Haell (bp)
263 205 157 106
ORB1 alleles 0401/0411 (!) 0403/0409 (!)
. +
+ -
0404/0411 (?) 0403/0410 (?)
- + + -
0408/0411 (V) 040310405 (V) 0407/0410 (V)
+
+
+
-
-
Combinations of
DRB1 alleles
+ +
58
48
+ + - + +
+ + + -
+ +
+
+
+
-
+
+ - + + + - + +
Figure 5. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRw52 associated antigen-DRBI genes obtained from 12 HTCs distributed at the Tenth International HLA Workshop. Determination of their genotypes was based on Table 10. The size o f the amplified region of the DRBl exon 2 is 265 bp as indicated.
Sacll+Hphl (bp) 120 109
72 71
62
58
+ +
- + + -
0403/0405 (V) 0407/0410 (V)
+ -
+
+ +
0403/0408 (.) 040410407 (.)
+ - +
+
+
+
+
-
+
47
+
-
11
+ +
+
+
Haell+Hphl. (bp) Combinations of DRBl alleles
0401/0410 (t) 0404/0409 (t) 0404/0405 (&) 0408/0410 (&) Combinations of
OR61 alleles 040110405 (#) 0408/0409 (#)
-: negative, +: positive.
192
120 109 106
95
72 71
14
11
+ + - + + + - + + + + + - - + + + + - + + - + + + +
113 107
+ - +
Haell+Mnll (bp) 79
78
71
35
+ + + + + + +
29
+
-
6
+
+
by 1 in Table Z), none in other alleles (indicated by 0 in Table 2) and both cleavage and non-cleavage sites for heterozygote (indicated by 2 in Table 2) in the amplified region by computer analysis and two different restriction endonucleases, AvaII and PstI were selected for digestion to detect allelespecific cleavage after PCR-amplification (Table 2). As shown in Table 2, these two different enzymes predict discrimination of 6 possible DR1DRBl allelic combinations with homozygote and heterozygote. Fig. 2 demonstrates that DRBl *(I101 (HOM2,9005) displayed the cleavaged patterns with AvaII (122bp, 84bp) and PstI (165bp, 41bp) and that DRB1*0102 (M2070782) had the cleavage site with AvaII (122bp, 84bp) but no cleavage one with PstI (206bp) as predicted from their nucleotide sequences. HTC of DRB1*0103 was not available in this study.
Modified PCR-RFLP for DRBl DR2-DRB1 genotyping
The DR2-DRBI genes were amplified with the use of the DRZspecific primers (S'R2 and 3'R: Table 1) from DRZpositive HTCs (Fig. 3). In a similar way, the nucleotide sequences of the DR2-DRB1 alleles were used to search for restriction endonucleases specific for each of the four DRZDRBI alleles by computer analysis, and 3 restriction endonucleases, FokI, Cfrl3I and HphI, were selected to detect allele-specific cleavage and RFLP patterns after PCR-amplification followed by enzyme digestion and found to produce the band patterns expected from Table 3 (Fig. 3). These 3 restriction endonucleases predict discrimination of all of 10 possible homozygous and heterozygous combinations by the DRZDRB1 alleles. D R b D R B l genotyping
Eleven subtypes of the DR4 antigen (DRB1*0401 to DRB1*0411) were determined by the modified PCR-RFLP method with 6 restriction enzymes listed in Table 4. After selective amplfication of the DR4-DRB 1 gene using the DR4-specific primers (5'R4 and 3'R). 11 subtypes can be typed by cleavage patterns with SacII, AvaII, HinfI and HaeII restriction enzymes and RFLP patterns with HphI and MnlI enzymes (26). All 66 possible combi-
nations (Tables 4, 5 and 6) of the DR4-DRB1 alleles in homozygous and heterozygous ways can be unequivocally discriminated with the restriction endonucleases selected here, except for nine combinations (DRB1*0403/0408 and 0404/0407, DRB1*0401/0411 and 0403/0409, DRB1*0404/ 041 1 and 0403/0410, DRB1*0408/0411 and 04031 0405, DRB1*0408/0411 and 0407/0410, DRBl*0403/0405 and 0407/0410, DRB1*0401/ 0410 and 040l0409, DRB1*0404/0405 and 0408/ 0410, DRB1*0401/0405 and 0408/0409) which give the identical cleavage and RFLP band patterns. As shown in Table 7, these heterozygotes, which cannot be discriminated by the PCR-SSO method theoretically (Fig. 4), can be distinguished by the use of double digestion (for example, for discrimination between SacII + HphI DRB1*0403/0408 and 0404/0407; see Fig. 4) in the PCR-RFLP method which allows examination of whether two polymorphic sites are located on the different chromosomes (trans position) or on the same chromosome (cis position). DRw52-associated (DR3, DRwl1, DRwl2, DRwl3, DRwl4 and DRw8) antigen-DRBI genotyping
Twenty-two suballeles of DR3, DRwl 1, DRwl2, DRwl3, DRwl4 and DR8 in the DRw52-associated group can be distinguished by the modified
Table a. Correlation between cleavage and RFLP patterns obtained by restriction endonucleases and DRB1 alleles for DR3,5.6 and w8 antigens Restriction Endonucleases Hphl DRBl alleles
0301 0302 1101 1103-4 1102 1201 1202 1301 1302 1303 1304 1305 1401 1404 1402 1403 1405
oaoi
0802 0804 0803
Group
Avall
Fokl
Kpnl
Haell
Cfrl31
sfaNl
Sacll
Bsall
Apal
3 3
A
1 0 0 0
1 1
C C 0
0 0 0
0 0
5u 1)
1 1 0 0 0 0 0 0 0 0 0 0 1
0
0
1 1
0 0 0 0 0 0 0 0 0
0 0
I 0
0 0 0 1 1 0
0 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 0 0 0 0 0 1 1 0 0 1
0 0 1 1 1 1 1 1 1 1 1 1
0 0 1 1 1 1 1 1
0 0 0 0 0 0 0
1
0 0
0 0
0 0 1
DR
5(11) 5U1) 5U2) 5(12) 6U 3) 6U3) 6 s 3) W3) 4 13) 6U 4) W4) 6U4) 6V4) 6U 4)
a a a a
B
E F G G H H I J J K L M N 0 0 P
0 1 1 0 1 1 1 1 0 0
1
, o
1
0 0
0 0
0
0 0 0 0 0
1 1
0 0 0 0
0
0 0 1
0
0 0 0 0 0 1 0 0 1
1 1
1 0 1
1 0
0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1
109 110
145
120
+ +
+
+ -
+ +
-
o
+ + + +
0 0 0
+ -
+ +
-
-
+
+ +
o
+ + -
0 1
0 1 1 1 1
0: not cleaved, 1: cleaved, +: positiie, -: negative.
193
Ota et al. Table 9. Cleavage patterns obtained by restriction enzymes in DRBl heterozygotes with DR3,5,6 and w8 alleles Combinations of DRB1 alleles
0301/0302 0301/110(1,3-4) 0301/I 102 0301/7201 0301/1202 0301/130(1,2) 0301/130(3,4) 0301/1305 0301/140(1,4) 0301/1402 0301/I 403 0301/1405 030110801 0301/080(2.4) 03011oao3 0302/110(1.3=4) 03031 102 03031201 03031202 0302/130(1,2) 0302/130(3,4) 03031305 0302/140(1,4) 0302/1402 0302/1403 030211405 03030801 0302/080(2.4) 0302/0803 110(1,3=4)/1102 110(1,3=4)/1201 110(1,3=4)/1202 110(1,3=4)/130(1,2) 11O( 1,3=4)/130(3,4) 110(1,3=4)/1305 110(1,3-4)/140(1,4) 110(1,3-4)/1402 110(1,3=4)11403 11O( 1,3=4)/1405 110(1,3=4)/0801 110(1,3-4)/080(2,4) 110(1,3-4)10803 1102/1201 1102/1202 1102/130(1,2) 1102/130(3,4) 1102/1305 1102/140(1.4) 1102/1402 110211403 1102/1405 110210801 1102/080(2,4) 1102/0803 1201I1 202 1201/130(1,2) 1201I130(3,4)
194
Restriction Endonucleases Groups
Avall
Fokl
Kpnl
Haefl
Crfl3l
SfaNl
Sacll
BsaJl
Apal
AiB
1 2 2 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 2 2 2 1 1 1 1 2 2 2 0 0 0 0 0 0 2 2 2 2 0 0 0 0
0 0 2 2 0 2 2 0 0 0 0 0 0 0 2 0 2 2 0 2 2 0 0 0 0 0 0 0 2 2 2 0 2 2 0 0 0 0
2 2 2
0 0 0
1 2 2
2
0 0 0 2 0 0
1 1 1 2 2
0 2 2 2 2 2 2 2
0 2 2 2 2 2 2 2 2
0 0 0 0 0 0 0 0 0
2
0
2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 0 2 2 2 0 0 0 0 0 0 0 0 0 2
A/C AID
AtE A/F
AIG AIH A/I A/J A/K AJL
AIM A/N
A/O AtP 0/c BID BIE
B/F BIG BIH 811 B/J B/K BIL B/M
BIN BIO
B/P C/D
C/E” C/F C/G*) CIH CII CIJ CIK CIL CIM” CIN CIO CIP’ D/E DIF” DIG OIH
0/l2’ DIJ DIK DIL DIM” D/N” D/O
O/P E/F
VG UH
0 0 0 0 2 2 2 2 0 0 0 0 0 0
0 0 0
2 1 2 1 1
2 2 2 2 2 2 2 1
2 1 1
2 2 2 2 2 2 2 2 2 2 2 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0
0 0
0 2 0 2 0 0 0 0 0 2 0 0 0 0 0
2 0 2 0 0 0 0 2 0 0 0 0 0 2 0 2 0 0 0 2 0 0 0 0 0 2 0 2
0 0 2
1
1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1
1 0 2 2
2 2 1 2 1 2 1 1 1 2 1 2 2 2 2 2 1 2 1 2 1 1 1 2 1
2 0 0 0 2 0 2 0 2 2 2 0 2 0 0 0
2 0
2 0 2 2 2 0 2 0 0 2 0
0
2 0 0 0 0 0 2 2 2 2 2 2 2 0 2 0 0 0 0 0 1 1
1 1 1
1 2
1 2 2 2 2 2 1
1 1 1 1 2 1 2 2 2 2 2 1 1 1
2
2 2 2 2 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
2 2 2 0 0 0
0 0 0 0 0 2 0
2 2 2 0 0 0 0 0 0 0
2 0 2 2 2 0 0 0
Modified PCR-RFLP for DRBl Table 10. RFLP patterns detected with Cfrl3I+Fokl, Fokl+SiaNI, Avall+SfaNI, Haell+ Fokl, Fokl+Apal and Sacll+Avall double digestion or Rsal digestion Cfrl31+Fokl (bp)
Combinations of
DRB1 allel6 110(1,3,4)/1201 1102/1202
DRB1 alleles
HIA-DRB1 genotyping from healthy Japanese
groups
265
201
170
CE DF
+
+
+ +
Combinations of groups
-
- Fokl+SfaNI (bp) 265
170
145
-
+
110(1,3,4)/130(1,2) 1102/1305
CG DI
i.
1201I1305 1202/130(1 ,Z)
El FG
-.
groups
265
181
145
CM IJ
+
+
+
~~
~
+
4-
+
+ -
-I-
+
Avall+SfaNI (bp)
DRB1 alleles 110(1,3,4)/1405 13051140(1,4)
__
Combinations of
Discussion
+
groups
265
170
159
110(1,3,4)/0803 1102/0801
CP ON
+
.-
+
120110801 1202/0803
EN FP
.-
+
.t
-
+ + + +
130(1,2)10801 130(3,4)/080(2,4) 1305/0803
GN
._
+-
HO
.t
IP
f
Combinations of
DRB1 alleles
groups
130(1.2)/0801 130(3,4)1080(2,4) 130510803
GN HO IP
Combinations of groups ~~~
IL KO
Combinations of
DRB1 alleles 130(3.4)/1403 140Z0803
+: positive, -: negative.
-
+ +
-
-
+
Fokl+Apal (bp)
265
205
170
+
+ + -
+ + +
-
DRBl alleles
11OZ1405 130(1,2)/140(1,4)
The HLA-DRB I genes which encode p chain molecules of DR antigens defined by the serological method, as well as Dw antigens recognized by
Haell+Fokl (bp)
DRB1 alleles
DRBl alleles
HLA-DRB 1 DNA typing was performed for genomic DNAs obtained from healthy Japanese volunteers. Generation of PCR-amplified bands with the DRB 1 group-specific primers indicated a complete agreement with DR antigens assigned serologically. Fig. 7 shows the heterozygous DRBl allelic patterns obtained by the modified PCR-RFLP method in 3 individuals (DR2/DR4; DRB1*1502/ 0401, DR4/DR6;DRBI*0405/1403, DR6/DR6; DRBI * 1303/ 1403).
~
Combinations of
130511403 1402/080(2,4)
except ones including DRBI* 1103 or 1104, as mentioned above.
-
207 ~
0 1 1 0
181
~
+ -
+
+
Rsal (bp)
150
140
111
OM GJ
+
4-
+ +
groups
111
101
HL KP
+
i.
groups
1
Sacll+Avall (bp)
265
+
1
+
81
+ -
2 2 0 2 1 1 2 2 2
Figure 7. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRBl exon 2 genes obtained from DNAs of 3 normal individuals (No. I; DRB1*1502/0401, No. 11; DRB1*0405/ 1403, No. 111; DRBI*I303/1403) after digestion with restriction enzymes. Their genotypes were determined on the basis of Tables 3, 4, 8 and 9.
197
Ota et al. Table 11. Patterns of polymorphic fragments detected with Hphl digestion, Fokl+Hphl or Sacll+Hphl double digestion for discrimination of heterozygotes between DRB1.1101 or 1103-4 allele and the other DR3,5,6 or w8-DRBl alleles Hphl (bp) Combinations of DRB1 alleles
145
120
+ + + + + + +
+ + + + +
030111101 03011110314 03081 101 03081103-4 110111101 110111103-4 1103=4/1103=4 110211101 110211103=4 1201/1101 120111103-4 1202/1101 120211103=4 1302/1101 130111103=4 130111101 130211103-4 130311101 1303/1103-4 1304/1101 1304/1103=4
+ + + + +
* *
A A
+ * *
B B
+ +
*
.
c
c
11
-
+
+
-
+
+
+ +
-
+ +
+
-
+ + +
-
+ +
+ + + -
+
+ +
+
+
+
-
+
-
+ +
+ + +
-
-
-
+ + + + + +
+ + + + + t
-
+ + + + + + +
+ + + + +
+ + + + -
-
-
+ + + + + -
+
+
+
+
+
+
-
+
+
+
+
080311101 080311103-4 ~
+ + + + + + +
+ + +
+ + + +
+ +
35
+ + + + - + + -
+ , + + + + +
130511101 130511103- 4 140111101 140W 10314 140411101 1404/1103=4 1402/1101 1402/1103=4 1403/1101 1403/1103=4 1405/1101 1405/1103=4 080111101 080111103=4 0802/1101 0802/1103=4 0804/1101 0804/1103=4
110 109
+ + +
-
+
+
+ + +
+
+
-
+ + +
+
+
+ + +
+ +
120
+
109
+
-
95
+ -
84
+
25
+ +
11
+ +
Fokl+Hphl (bp) (*B)
145
+ +
120
+ -
109
95
+
+
-
-
84
25
11
+
-
+ +
+ +
58
47
35
+
+ +
+ +
+
Fokl+Hphl (bp) (.A)
145
-
+ -
+
+
+
Sacll+Hphl (bp) (.C)
145
+
+ -
+ +
120
+
110
+ +
62
+ +
+ -
+
+ +
11
+ +
~
+: positive, -: negative. Three pairs of *A, 08and 4 can be discriminated by RFCP band patterns obtained after double digestion with Fokl+Hphl and Sacll+ Hphl, respectively.
cellular typing, became assignable for a high degree of their polymorphisms, type at the nucleotide level using PCR procedures such as PCR-SSO typing with raido-labelled oligonucleotide probes (27-28) and with non-radioactive oligonucleotide probes (29). However, the PCR-SSO method requires multiple SSO probes, especially for the DRBI genes with as many as 43 allles, as well as labeling and washing temperatures with several stringencies 198
(27-33). A non-radioactive “reverse dot blot” method has also been used for SSO typing (15), but the amount and length of oligonucleotide probes must be adjusted and suitable conditions for hybridization determined, which is technically demanding. In this study, in order to establish a simple and practical PCR-DRB 1 genotyping, the modified PCR-RFLP method which depends mainly on cleavage or no cleavage of the amplified
Modified PCR-RFLP for DRBl Table 12. Patterns of polymorphic fragments detected with Hphl digestion, Haell+Hphl and Sacll+Hphl double digestion for discrimination of heterozygotes between DRB1.1301 or 1302 allele and the other DR3,5,6 or wBDRB1 alleles HDhl (bo) Combinations of DRB1 alleles
030111301 0301/1302 0302/1301 0302/1302 11OZ1301 11OZ1302 12011'1301 1201/1302 120Z1301 1202il302 1301/1301 1301/1302 1302/1302
145
120
110 109
35
11
+ + + +
+ + + -
+ +
-
-
+ + + + + , + + + + + + +
+ +
+
+
+
1303/1301 1303/1302 13041301 1304/1302
*
A
.
A
+
+
1305/1301 1305/1302 1401/1301 140111302 1404/1301 1404/1302 1402/1301 1402/1302 1403/1301 140311302 1 40511301 1405/1302 0801/1301 0801/1302
*B
0803/1301 0803/1302 ~~
+: positive, -: negative. Two pairs of *A
+
+
+
+ + + + + + + + + + + +
+ + + + + + + + + -
-
-
+
-
+
+ + + + + + + + + +
+ -
+ + + + + +
+ + + -
+ + +
-
+ -
-4
+
-
+ -
-
+
-
+
+
+
+ + + + +
+
+
+
+
.B
0802/1301 0802/1302 0804/1301 0804/1302
+ + -
+
-
+
+
+
+
+ + +
-
+
+
-
-
-
-
-
-
+ + + + +
+ + + +
+
~
+
+ + + + + + + + + + + + + + +
+ + +
145
120
+
-
145
120
+
+
+
+
Haell+Hphl (bp) (.A) 109 106 95
+
-
14
11
+
+
Sacll+Hphl (bp) (4) 110 62 58 47
+
+
+
-
35
11
+
+
-
+
+
+
-
-
+
+
+
-
+
+
+
+
+ ~~
~
~~~~~
and *B can be discriminated by RFLP band patterns obtained after double digestion with Haell+Hphl and Sacll+
Hphl, respectively.
segment by informative restriction enzymes was applied to the DRBl gene. This modified PCRRFLP method also utilizes allele - or group-specific primers in order to avoid cross hybridization with other DRBZ-DRBS genes and can distinguish all the 43 previously described alleles to date differing in amino acid sequence in the PI domain in homozygous and heterozygous ways, except for DRB1*1103 and 1104. The PCR-RFLP method is a simple, practical and reproducible technique for accurate definition
of HLA alleles without the use of multiple probes, and their labeling and can clearly discriminate even one base difference between alleles by restriction enzyme. Further, one of the advantages over the PCR-SSO method is that PCR-RFLP can show linkage between two polymorphic sites of interest by measurement of the length of a fragment generated after double digestion with two restriction enzymes (cis or trans position, Fig. 4), while the PCR-SSO cannot provide any such information. A characteristic feature of genetic polymorphisms 199
Ota et al. Table 13. Patterns of polymorphic fragments detected with Hphl digestion, Hphl+Sac11 double digestion for discrimination of heterozygotes between ORB1+1303or 1304 allele and the other OR 3,5.6 or w8-ORB1 alleles Hphl (bP) Combinations ot DRBl alleles
0301I1 303 0301I1304 0302/1303 030U1304 11 02/1303 11 02/1304 120111303 1201I1 304 120U1303 120U1304 1303I1303 1303/1304 130411304 1305I1303 1305/1304 1401I1 303 140111304 1404/1303 1404/1304 1402/1303 1402I1304 140311303 140311304 140511303 140511304 080111303 0801I1304
145
120
110 109
35
11
+
+
+
-
+
+
-
+
+ + +
+ +
+
-
+
+
+
+
+
-
+
+
+
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + -
+ + + + + + + + + + + + + + -
+
+
-
+ , +
-
0802/1303 0802I1304 0804/1303 0804/1304 0803/1303 0803/1304
+: positive, +: strong positive, -:
+
* *
A A
+
-
+ +
+ +
+
+ +
-
-
+ +
-
-
+ + + + +
+ + + +
-
+ + +
-
-
+ + -
+
+ +
+ +
+ + + + + + +
+
+
+
+ +
+
+ +
+ +
+
+ +
-
Sacll+Hphl (bp) CA)
145
+ +
120
+
110
+ +
62
+ +
58
+ -
47
+
35
11
+
+
+
+
+ +
negative, pair of *A can be discriminated by RFLP band patterns obtained after double digestion with Sacll+Hphl.
in the HLA genes is that they generally have no allele-specific nucleotide sequences, but that alleles differ in combinations of variable regions by taking patchwork structure. Therefore,. 15 pairs of heterozygotes* so far examined in the DRBI gene, with the same combinations of variable regions, result in the same reactive patterns to SSO probes in the PCR-SSO method as in the case of DRB1*0403/ 0408 and DRB1*0404/0407 in Fig. 4. In contrast, in the PCR-RFLP method double digestion gives *I DRB1*0401/0411 and 0403/0409, DRB1*0404/0411 and 0403/0410, DRB1*0408/0411 and 0403/0405, DRB1*0408/ 041 1 and 0407/0410, DRB1*0403/0405 and 0407/0410, DRB1*0403/0408 and 0404/0407, DRB1*0401/0410 and 040410409. DRB1*0404/0405 and 0408/0410, DRB1*0401/ 0405 and 0408/0409, DRB1*1101/1301 and 1104/1302, DRB1*0804/1101 and 0802/1104, DRB1*0892/1301 and 08041 1302, DRBl *I 103I 1201 and 1 102/ 1202, DRBl*0801 / 1102 and 0803/1103, DRB1*0801/1201 and 0803/1202.
200
-
the information on the linkage between two polymorphic sites by the appearance of the cleaved fragments which allow unequivocal discrimination between such heterozygotes. Incomplete or partial digestion of the PCR products by restriction enzymes, which will obscure definite assignment of HLA alleles can be overcome by preparation of positive control DNAs for ensuring that the digestion is complete. Further, PCR primers incorporating recognition sites for recognition enzymes used here will be more useful as an internal control. Unusual patterns of cleavage by restriction enzymes virtually indicate the presence of new alleles in the HLA genes, which must be confirmed by determination of their nucleotide sequences. This modified PCR-RFLP method could also be successfully applied to complete DRB 1, DQAl ,
Modified PCR-RFLP for DRBl Table 14. Patterns of polymorphic fragments detected with Hphl digestion, Apal +Hphl double digestion for discrimination of heterozygotes between DRBl.1401 or 1404 allele and the other DR 3,5,6 or w8-DRBl alleles Hphl (bp) Combinations of DAB1 alleles 0301/1401 0301/1404 030211401 030211404 110211401 110211404 1201/1401 120111404 120211401 120211404 130511401 1305/1404 140111401 1401/1404 140411404 1402/1401 140211404 140311401 1403/1404 140511401 140511404 0801/1401 0801I1404
145
120
110 109
+
+ +
+ + +
+
+ + + + + ' + + + + +
+ + + +
I+ . ++ +
+ +
-
+ + + + +
+ + + + + + + -
+
+ + +
+ + + + + + +
-
+ + + +
+ + + + + + + + +
35
11
-
-
+ +
-+
+ +
+ + + + + +
+ -
-
+ + -
+ + + + + + Apal+Hphl (bp) 1.44)
0802/1401 0804/1401 080211404 0804/1404 0803/1401 080311404
*A *A *B *B
+ + + -
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
145
+ + -
120
+ + + +
110
+ + + +
60
+ + + +
49
+ + -
35
+ + + +
11
+ + -
+: positive, +: strong positive, -: negative, pairs of *A and *B can be discriminated by RFLP band patterns obtained after double digestion with Apal+Hphl.
and DQB 1 genotyping as reported previously (22-23). This genotyping analysis can be made easily accessible to automation with a personal computer program for identification of HLA alleles. Further, PCR-RFLP is much cheaper than PCRSSO typing, especially for small numbers of samples. Thus, the PCR-RFLP method will be substituted for conventional serological and cellular typing and a good alternative to the PCR-SSO method. Acknowledgments
We thank Dr. J. Trowsdale of Imperial Cancer Research Fund for critical reading of the manuscript. This work was supported by a grant-in-aid for scientific research in 1991 from the Japanese Ministry of Education.
References 1. Bach FH. Class I1 genes and products: HLA-D region. Imrnunol Toby 1985: 6: 89-94. 2. Zinkernagel RM, Doherty PC. Restriction of in vitro T
3. 4.
5.
6. 7.
cell mediated cytotoxicity in lymphocytic choriomeningitits within a syngenic or semiallogenic system. Nature 1974: 248: 701-3. Babbit BP, Allen PM, Matsueda G, Haber E, Unanue ER. Binding of immunogenic peptides to Ia histocompatibility molecules. Nature 1985: 317: 359-61. Buus S, Sette A, Colon S, Miles C, Grey HM. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science 1987: 235: 1353-8. Guillet JG, Lai MZ, Briner TJ, et al. Immunological self/ nonself discrimination. Science 1987: 235: 865-70. Schwartz RH. T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Ann Rev Immunol 1985: 3 237-50. Bach F, Sachs DH. Transplanted immunology. N Engl J Med 1987: 317: 489-92.
201
Ota et al. Table 15.
Patterns of polymorphic fragments detected with Hphl digestion for discrimination of heterozygotes between DRB7.0802 or 0804 allele and the other DR 3,5,6 or w8-DRB1 alleles Combinations of DRB1 alleles
0301/0802 0301/0804 0302/0802 0302/0804 1102/0802 1102/0804 1201/0802 1201/0804 1202i0802 1202/0804 1305/0802 1305/0804 1402/0802 1402/0804 1403/0802 1403/0804 1405/0802 1405/0804 0801/0802 0801/0804 0802/0802 oao2/oao4 0804/0804 0803/0802 0803/0804
Hpbl (bp)
145
120
110 109
35
11
+ +
+
+ + + + + +
+ -
+ i.
+ + + + +
-
-I.
+ -
+
+ + -
+
+ + +
+ + +
+ + + ' + + + + + + + + + + + +
+
+ +
+
+ + +
+ + + + + + + + + +
+
+ + + + + +
+ +
+
-
+
+ -
+
+ + + + + + -
+
+ + + + +
+: positive, -: negative. 8. Khagani A, Yacoub M, McCloskey D, et al. The influence of HLA matching, donor, recipient sex, and influence of acute rejection on survival in cardiac allograft ren'pients receiving cyclosporin A and Azathioprine. Transplant Proc 1989: 21: 799-800. 9. Todd JA, Acha-Orbea H, Bell JI, et al. A molecular basis for MHC class 11-associated autoimmunity. Science 1988: 240: 1003-9. 10. Marsh SGE, Bodmer JG. HLA class I1 nucleotide sequences. Tissue Antigens 1991: 37: 181-9. 1I . Marsh SGE, Bodmer JG. DR and DQ epitopes and monoclonal antibody specificity. Immunol Today 1990: 1 0 305-1 I. 12. Grosse-Wilde H, Doxiadis I, Brandt H. Definition of HLAD with HTC. In: Albert MT, Baur MT, Mayr WR, eds. Histocompatibility Testing 1984. Heidelberg: Springer-Verlag, 1984: 249-64. 13. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of P-globin genominc sequence and restriction site analysis for diagnosis of sickle cell anemia. Science 1985: 230: 1350-4. 14. Mullis KB, Fallona F. Specific synthesis of DNA in vitro via polymerase catalyzed chain reaction. Methodr Enzymol 1987: 155: 335-50. 15. Saiki R, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequencespecific oligonucleotide probes. Proc Natl Acad Sci USA 1989: 86: 6230-4. 16. Trucco G, Fritsch R, Giorda R, Trucco M. Rapid detection of IDDM susceptibility with HLA-DQ-alleles as markers. Diabetes 1989: 38: 1617-22.
17. Yunis I, Salazar M, Yunis EY. HLA-DR generic typing by AFLP. Tissue Antigens 1991: 38: 78-88. 18. Maeda M, Murayama H, Ishi H, et al. A simple and rapid method for HLA-DQAl genotyping by digestion of PCRamplified DNA with allele specific restriction endonucleases. Tissue Antigens 1989: 3 4 290-8. 19. Maeda M, Uryu N, Murayama H, et al. A simple and rapid method for HLA-DP genotyping by digestion of PCRamplified DNA with allele-specific restriction endonucleases. Human Immunol 1990: 27: 111-21. 20. 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 specific restriction endonucleases. Tissue Antigens 1990: 3 5 20-31. 21. Olerup 0. HLA-class I1 typing by digestion of PCR-amplified DNA with allele-specificrestriction endonucleaseswit1 fail to unequivocally identify the genotypes of many homozygous and heterozygous individuals. Tissue Antigens 1990: 36 83-7. 22. Nomura N, Ota M, Tsuji K, Inoko H. HLA-DQBI genotyping by a modified PCR-RFLP method combined with group-specific primers. Tissue Antigens 1991: 38: 53-9. 23. Ota M, Seki T,Nomura N, et al. Modified PCR-RFLP method for HLA-DPB1 and -DQAI genotyping. Tissue Antigens 1991: 38: 60-71. 24. 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. 25. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymaticamplification of DNA with a thermostable DNA polymerase. Science 1988: 239: 487-9 I. 26. Ota M, Seki T, Kiyosawa K, et al. A possible association between basic amino acids of position 13 of DRBl chains and autoimmune hepatitis. Immunogenefics 1992 (in press). 27. Vaugham RW, Lanchburg JSS, Marsh SGE, Hall MA, Bodmer JG, Welsh KI. The application of oligonucleotide probes to HLA class I1 typing of the DRB subregion. Tissue Antigens 1990: 36: 149-55. 28. Mach B, Tiercy J-M. Genotypic typing of HLA class 11: from the bench to the bedside. Human Immunol 1991: 30: 278-84. 29. Scharf SJ, Griffith RL, Erlich HA. Rapid typing of DNA sequence polymorphism at the HLA-DRBI locus using the polymerase chain reaction and nonradioactive oligonucleotide Drobes. Human Immunol 1991: 30: 190-20 1. 30. Gao X,.Moraes JR, Miller S, Stastny P. DNA typing for class I1 HLA antigens with allele-specific or group-specific amplification V. Typing for subsets of HLA-DR1 and DR'Br'. Human Immunol 1991: 30: 147-54. 31. Moraes ME, Vina MF, Stastny P. DNA typing for class I1 antigens with allele-specificor group-specificamplification. IV. Typing for alleles of the HLA-DR2 group. Human Immunol 1991: 31: 139-41. 32. Gao X, Vina MF, Shumway W, Stastny P. DNA typing for class I1 HLA antigens with allele-specific or group-specific amplification. 1. Typing for subsets of HLA-DR4. Human Immunol 1990: 2 7 40-50. 33. Petersdorf EW, Smith A, Mickelson EM, Martin PJ, Hansen JA. Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRBl first domain. Immunogenefics 1991: 33: 26.5-71. 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: 0263-34-8480
Introduction The HLA class I1 genes (HLA-DR, -DQ, -DP), located in the short arm of chromosome 6, specify heterodimeric glycoproteins involved in the regulation of the immune response (1). These highly polymorphic molecules bind to foreign or self antigenic peptides and present them to antigen-specific T cells in a self-restricted fashion (2-5). Accurate identification of their polymorphisms is a prerequisite for determination of the functional role of HLA in immunoreactivity (6) and transplantation immunity (7-8), as well as in the susceptibility to autoimmune diseases (9). In the DRB region, five DRB (DRB1, DRB2, DRB3, DRB4 and DRB5)
Masao Ota', Takeshi Seki', Hirofumi Fukushima', Kimiyoshi Tsuji' and
Htdstoshi Inoko' 'Department of Legal Medicine, *Second Department of Internal Medicine, Shinshu University School of Medicine, Nagano. and 'Department of Transplantation, Tokai University school of Medicine, Kanagawa, Japan
Key words: PCR: polymerase chain reaction RFLP: restriction fragment length polymorphism - SSO: sequencespecificoligonucleotide - DRBl alleles - genotyping Received 16 September, revised, accepted for publication 4 December 1991
loci have been identified and these DRB genes, except for a pseudogene, DRB2, are known to encode specific DR chain molecules. Among them, the DRBl genes is thought to be the most important in controlling the immune response as well as the main alloantigen determinants. There are now as many as 46 DRBl alleles known,among which 43 have different amino acid sequences in the /3 1 domain-exon (10). DR specificities are usually determined by serological procedures using alloantisera or monoclonal antibodies. The availability of these antibodies is limited in specificity, quantity and sensitivity for full DRB typing (1 1). Furthermore, the cellular assay procedure (12) used for definition of Dw specificity is time-consuming and 187
Ota et al. Table 1. PCR primers for allele- or groupspecific amplification of the DRBl gene ~
Gene DRBlS primer
DRBl 3’ primer
Primers for DR2 for DR4 for DR9 for DRl forDR7 for DR10 DR3
5’R2 5‘R4 539-1 5’Rl SR7 SR10
for DR6 DR5 DR8
5’R3568
3’R
codon matched
Sequences (5’ to 3’)
PCR-products
7-1 3 6-1 3 8-1 5 25-32 25-32 25-32
TTCCTGTGGCAGCCTAAGAGG GmCTTGGAGCAGGllAAAC GPAGCAGGATAAGmGAGTG GGTTGCTGGAAAGAECATCT AGTTCCTGGAAAGACTCTTCT GGTGCTGGAAAGACGCGTCC
(261 bp) (263bP) (256bP) (206bp) (206bp) (206bP)
5-1 2
ACGmCTTGGAGTAClCTACG
(265hp)
87-93
complicated by the limited availability of the typing reagents, homozygouS typing cells (HTCs). Restriction fragment length polymorphism (RFLP) analysis with a genomic Southern hybridization technique allows the identification of DRB alleles at the gene level, but requires a large amounts of high-molecular weight DNAs and a complicated procedure; also, the interpretation of RFLP band patterns is not straightforward. The limitation of these methods for identification of DRB polymorphisms has been overcome by the advent of polymerase chain reaction (PCR) technique
CCGCTGCACTGTGAAGCTCT
(13-17). The PCR method permits precise and direct analysis of allelic variations with as little as lng of genomic DNA. The PCR-RFLP method we reported previously (18-20) 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 the majority of heterozygotes cannot be discriminated from each other (21). These problems have been overcome by the modified PCR-RFLP method of incorporating informative restriction enzymes which have a single recognition site in some alleles, but none in other alleles in the amplified segments for the DPB1, DQAl and DQB1 genes (22-23) and thus reading of RFLP band patterns generated has become much simpler and easier. In this study, this modified PCR-RFLP method was applied to DRBl genotyping along with 7 pairs of group-specific primers. The PCRamplified DNAs digested with restriction endonucleases (AvaII and PstI for DR1 group; FokI, Cfrl3I and HphI for DR2 group; SacII, AvaII, HinfI, HaeII, HphI and MnlI for DR4 group; AvaII, FokI, KpnI, HaeII, Cfrl31, SfaNI, SacII, BsaJI, ApaI, HphI and RsaI for DR3,5,6 and 8 group) were subjected to electrophoresis. DRBl Table 2. Correlation between cleavage patterns obtained by two restriction endonucleases and DRB1 homozygous and heterozygous alleles for OR1 Combinations of
Figure 1. The PCR products were amplified by each pair of the group-specific S’primers (SR1, S’R10, SR2, S’R4, S’R7, SR9 and S‘R3.568) and common 3’(3’R) primer. DNAs were extracted from HTCs and a healthy donor cells having DRwlOIDR2 heterozygote as indicated.
188
Restriction endonucleases
DRBl alleles
Groups
Avall
Pstl
0101/0101 0102/01 02 0103/0103 010110102 010110103 0102/0103
NA
1 1 0
1 0
BIB
c/c
A/B NC BiC
1
1 2
2 2
1 2
0: not cleaved, 1: cleaved, 2: both for heterozygote.
Modified PCR-RFLP for DRBl International Histocompatibility Workshop. They were isolated from EBV-transformed HLA homozygous B lymphoblastoid cell lines which were the same ones used in the previous study (23). Genomic DNAs from healthy Japanese volunteers were isolated by phenol extraction of sodium dodecyl sulfate (SDS)-lysed and proteinase K-treated cells, as described earlier (24). As no homozygous DRwlO cell line was available, a DRwlO heterozygote (DR2/DRwlO) was included for obtaining the standard RFLP band pattern of DRwlO.
Table 3. Cleavage and RFLP patterns obtained by three restriction endonucleases in combinations of DRBl homozygous and heterozygous alleles for OR2 specificity
-~ ~
Combinations of
Restriction endonucleases Hphl (bp)
ORB1 alleles
Groups
Fokl
Cfrl31
120
109
150111501 1502/1502 1601I1601 1602/1602 1 5 0 1502 ~ 1501I1601 1501I1602 1502/1601 1502/1602 1601/1602
NA BIB C/C
1 1 0 0 1 2 2 2 2 0
1 1 0 1 1 2 1 2 1 2
+
+ +
~~
DID
A/B NC NO 6IC BID C/D
+
+ +
-
-
+ + + + +
PCR amplification
Genomic DNA (200-300 ng) was amplified by the PCR procedure with 2.5 units of the Taq DNA polymerase (Perkin Elmer Cetus Inc.) (25). The reaction mixture (genomic DNA, PCR buffer; 50 mM KCl, 2.5 mM MgCl,, 10 mM Tris-HC1 pH8.4, 0.01% gelatin, 0.02% N(Nonidet)P-40; 200 pM each of dATP, dCTP, TTP and dGTP; 1 pM of each of the primers) and distilled water for a total volume 100 pI in a 1.5 ml eppendorf tube (Eppendorf Co.) was covered with 50 pl of mineral oil to prevent evaporation and was subjected to 30 cycles of 1 min at 94'C, I min at 6OoC, and 2 min at 72°C by automated PCR thermal sequencer (Iwaki Glass Inc.). For DR1 and DR9 amplification, annealing was performed at 55°C. The second exon of the DRBl gene was amplified using 1 of 7 groupspecific 5' primers (5'R2,5'R4,5'R9- I , 5'R 1, YR7, SR10 and YR3568) along with the common 3' primer (3'R) as shown Table 1.
+
+
~~
0: not cleaved, 1: cleaved, 2: both for heterozygote. +: positive, -: negative.
genotypes were determined mainly by checking whether the amplified DNAs are digested or not. Heterozygotes could also be analyzed with the method. This modified PCR-RFLP method resolves the multiple subtypes within the major DR groups and is technically simpler, cheaper and more practical for routine HLA typing work than our previous PCR-RFLP method, and thus is a good alternative and a complementary technique to the PCR-SSO method.
Material and Methods DNA samples
Seventy DNA samples used in this study were distributed for Southern blot analysis in the Tenth
Table 4. Cleavage and RFLP patterns obtained by six restriction endonucleases in DR4-DR61 hornozygous alleles Restriction endonudeases
Group
Hphl (bp) DR4-DRB1 alleles Sacll Avall Hinfl Haell 120 109
B
0
-
+
+
-
O
+
-
-
+
1 1 1
0 0 0
0
1
0
0
0
0403 0407
0
1 1
0 0
0 0
+
0
0405 0409 0410
1 1 1
1 1 1
0
1 1 1
-
+
0 0
-
+
+
-
+
-
-
+
0406
0
1
1
0
0
1
0
1
0402 ~
1 1
1 0
DRBM3101/0101 oRBrolo2/0102
HOM2
M2070782
Figure 2. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRI-DRBl genes obtained from DNAs of two HTCs after digestion with AvaII and PstI enzymes. Determination of their genotypes was based on Table 2. The size of the amplified region of the DRI-DRBI exon 2 is 206 bp as indicated.
C ~~
-
+
E F ~
0411 ~~~
-
+
- +
~~~
0
113 107
1 1 1
0401 0404 0408
A
Mnll (bp)
- +
~~
0:not cleaved, 1: cleaved, +: positive, -: negative.
189
Ota et al.
Digestion with restriction endonucleases After amplification, aliquots (7 pl} of the reaction mixtures were digested with restriction endonucleases (AvaII and PstI for DRl-DRB1; FokI, Cfrl3I and HphI for DR2-DRB1; SacII, AvaII, HinfI, HaeII, HphI and Mn1I for DRCDRBl; AvaII, FokI, KpnI, HaeII, Cfrl31, SfaNI, SacII, BsaJI, ApaI, HphI and RsaI for DR3,5,6 and 8DRB1 and 1-2 units) for 1 to 3 h after addtition of appropriate incubation buffer at suitable temperature. Complete digestion of restriction enzymes was confirmed by inluding positive control DNAs with the HLA alleles which have cleavage sites for respective enzymes in the PCR-amplified regions.
Table 5. Cleavage patterns obtained by restriction enzymes in combinations of DR4DRBI heterozygous alleles Combinations of
0403 (C) 0407 0401 0404 0408
0402 (B)
0403 (C)’ 0407
1 1 1 1 I
1
0 0
Fokl
1 1 1 1 1 1
Restriction endonodeases
DRB1 alleles Sacll
Avall
Hinfl
Haell
2
1
0
0
0405 0409 (0) 0410 0406 (E)
1
1
0
2
2
1
2
0
0411 (F)#
2
1
0
2
0403 (C) 0407
2
2
0
0
0
2
0405 0409 to) 0410 0406 (E)
1
2
2
2
2
0
0411 (F)
2
2
0
2
0405 0409 (D)# 0410
2
1
0
2
0406 (E)
0
1
2
0
0411 (F)
0
1
0
2
0405 0409 (0)’ 0410
0406 0411 (F)
2 2
1
2
2
1
0
1
0406 (E)
0471 (F)
0
1
2
2
0:not cleaved, 1: cleaved, 2: both for heterozygote. (Cr 0403,0407and (Dy 0405,0409,0410can be (A)’ 0401,0404,0408, distinguished by the use of Hphl and Mnll restriction enzymes (Table 6), or Sacll+Haell, Sacll+Hphl, Haell+Hphl or Haell+Mnl I double-digestion technique (Table 7). Combinations (#) of AF (0401,0404 or 0408/0411)to CD (0403 or 0407/0405.0409 or 0410) can be discriminated from each other by RFLP band patterns obtained after Hphl and.Mnll enzymes [Table 6), or Sacll+Hphl or Sacll+Haell double digestion (Table 7).
0 1
Cfrl31
Acrylarnide 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.). Cleavage or no cleavage of amplified fragments was detected by staining with ethidium bromide.
Hphl Figure 3. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRZ-DRBI genes obtained from DNAs of 8 HTCs after digestion with FokI, Cfrl3I and HphI enzymes. Determination of their genotypes was based on Table 3. The size of the amplified region of the DRZDRBI exon 2 is 261 bp as indicated.
190
Results Groupspecific amplification Fig. 1 shows the products of PCR amplification by 7 pairs of the group-specific 5’ primers and common 3’ primers (Table 1). Twelve genomic
Modified PCR-RFLP for DRBl Table 6. Patterns of polymorphic fragments detected with Hphl and Mnll enzymes for discrimination of heterozygotes between DRB1*0401, 0404 or 0408. DRB1’0403 or 0407, or DRB1.0405, 0409, or 0410 and other DACDRBl alleles
0401 0404 0401 0404 0408
Mnll (bp)
Hphl (bp)
Combinations of DRBl alleles
109
+ + + +
0401 0404 0408 0408
-+
0402
0401 0404 0408
0403
0401 0404 0408
0407
0401
0405 # 0409 0410 $
0404
0405 & 0409 S 0410
0408
0405 0409 # 0410 &
0401 0404 0408
0406
0401 0404 0408
+ +
-
+ + +
+ ~~
~~
+ +
120
107
113
-
-
+
+ + +
+ + +
-
+ ++ + +
+
+ -
+
~~
-
*
-
-
+
-
+ + + +
+
+ + -
-
+
+
+ + + + + + -
+ + +
+ + + +
+
+ + + +
+
! 0411 ? Y
+ -
+ +
+ +
0403
0405 Y 0409 ! 0410 ?
0407
0405 0409 0410 V
+ + + + + +
+ + + -
+ + + +
0402
0403 0407
+
+
+
+ + +
+ +
+
+
+
+
+
+ +
+
-
-
+ +
+
-
+ + + + -
-
-
+ +
0403
0407
+
.f
+ + +
0403 0407
0406
-
.t
+
0403 0407
0411
.f
+
+
+
+
+
-
0405 0409 0410
+
-
+
0402
+ -
+
+ + + +
+
-
-
+
+ -
Cont. table 6. Hphl (bp)
Combinations of DRB1 alleles 0405 0409
Mnll (bp)
109
120
107
113
0409 0410
+
-
+ +
+ -
0410
+
+
+
+
+
+
-
+
+
~~
+
0405 0409 0410
0406
0405 0409 0410
0411
~~~
+
+ + + + +
+ + -
~~~
~
+ + + + +
+ -
+ -
~~
-: negative, +: positive. Combinations of 0401/0411 and 0403/0409 (!), 0404/0411 and 0403/0410 (?), 0408/0411 and 0403/0405 or 0407/0410 (V) can be distinguished by RFLP bands obtained by doubledigestion with Sacll+Haell. Combinations of 0403/0405 and 0407/0410 (V) and 04031 0408 and 0404/0407 [‘) can be distinguished by doubledigestion with Sacll+Hphl. Combinations of 0401/0410 and 0404/0409 (S)and 0404/0405 and 0408/0410 (a) can be distinguished by doubledigestion with Haell+ Hphl. Combination of 0401/0405 and 0408/0409 (#) can be distinguished by doubledigestion with Haell+Mnll (See Table 7).
DNAs were extracted from HTCs distributed at the Tenth International HLA Workshop (HOM2,9005 on lane 1; WT8,9017 on lane 3; KASOII,9009 on lane 4; SAVC,9034 on lane 5; MOU,9050 on lane6; DXB,9075 on lane 7; VAVY,9023 on lane 8; BM21,9043 on lane 9; BM15,9040 on lane 10; OMW,9058 on lane 11 and TEM,9057 on lane 12) and healthy donor cells having DRwlO/ DR2 heterozygote on lane 2. These DRBl group-speficific primers (5’R2 to 5’R3568) were confirmed as amplifying the DRBl genes selectively from the DR2, DR4, DR9, DR1, DR7, DRw 10 and DRw52-associated antigens (DR3,
*
: Hphl
7 :S a d
Figure 4. The PCR-RFLP method can show linkage between two polymorphic sites. The nucleotide sequences are identical between DRB1*0403 and 0407 and between D R B l * W and 0408 except at the HphI(*) (in the right end) and Sac11( V ) sites. A characteristic feature of genetic polyrnorphisms displayed by the HLA genes is that their alleles can be distinguished by differing in combinations of variable regions by taking patchwork structure in the first domain exon. In the PCR-RFLP method double digestion (in this case, HphI+SacII) gives the information on the linkage between two polymorphic sites (here, the Sac11 and righ-end HphI sites) by the appearance of the cleaved fragment (in this case, a 47 bp fragment) which allows unequivocal discrimination between such heterozygotes.
191
Ota et al. DR5, DR6 and DR8), respectively. The DR7, DR9 and DRwlO alleles which have no suballele (DRB1*0701 and 0702 have the same nucleotide sequences in their j3l domain exons) can be typed simply by the presence of amplified bands generated with these group-specific primers (more exactly, allele-specific primers in these cases) as DRBl*O701 or 0702, 0901 and 1001, respectively. DR1-DRB1 genotyping
The DNA sequence allelic variations in the 206bp PCR-amplified region of the j3l domain exon in the D R l -DRB 1 genes (lo), where polymorphism in the DR 1-DRB 1 locus is primarily localized, were used to search for restriction' endonucleases which have a single cleavage site in some alleles (indicated Table 7. RFLP patterns detected with Sacll+Haell. Sacll+Hphl, Haell+Hphl or Haell+ Mnll doubledigestion for discrimination among 9 pairs of DR4 heterozygotes Combinations of
Sacll+Haell (bp)
263 205 157 106
ORB1 alleles 0401/0411 (!) 0403/0409 (!)
. +
+ -
0404/0411 (?) 0403/0410 (?)
- + + -
0408/0411 (V) 040310405 (V) 0407/0410 (V)
+
+
+
-
-
Combinations of
DRB1 alleles
+ +
58
48
+ + - + +
+ + + -
+ +
+
+
+
-
+
+ - + + + - + +
Figure 5. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRw52 associated antigen-DRBI genes obtained from 12 HTCs distributed at the Tenth International HLA Workshop. Determination of their genotypes was based on Table 10. The size o f the amplified region of the DRBl exon 2 is 265 bp as indicated.
Sacll+Hphl (bp) 120 109
72 71
62
58
+ +
- + + -
0403/0405 (V) 0407/0410 (V)
+ -
+
+ +
0403/0408 (.) 040410407 (.)
+ - +
+
+
+
+
-
+
47
+
-
11
+ +
+
+
Haell+Hphl. (bp) Combinations of DRBl alleles
0401/0410 (t) 0404/0409 (t) 0404/0405 (&) 0408/0410 (&) Combinations of
OR61 alleles 040110405 (#) 0408/0409 (#)
-: negative, +: positive.
192
120 109 106
95
72 71
14
11
+ + - + + + - + + + + + - - + + + + - + + - + + + +
113 107
+ - +
Haell+Mnll (bp) 79
78
71
35
+ + + + + + +
29
+
-
6
+
+
by 1 in Table Z), none in other alleles (indicated by 0 in Table 2) and both cleavage and non-cleavage sites for heterozygote (indicated by 2 in Table 2) in the amplified region by computer analysis and two different restriction endonucleases, AvaII and PstI were selected for digestion to detect allelespecific cleavage after PCR-amplification (Table 2). As shown in Table 2, these two different enzymes predict discrimination of 6 possible DR1DRBl allelic combinations with homozygote and heterozygote. Fig. 2 demonstrates that DRBl *(I101 (HOM2,9005) displayed the cleavaged patterns with AvaII (122bp, 84bp) and PstI (165bp, 41bp) and that DRB1*0102 (M2070782) had the cleavage site with AvaII (122bp, 84bp) but no cleavage one with PstI (206bp) as predicted from their nucleotide sequences. HTC of DRB1*0103 was not available in this study.
Modified PCR-RFLP for DRBl DR2-DRB1 genotyping
The DR2-DRBI genes were amplified with the use of the DRZspecific primers (S'R2 and 3'R: Table 1) from DRZpositive HTCs (Fig. 3). In a similar way, the nucleotide sequences of the DR2-DRB1 alleles were used to search for restriction endonucleases specific for each of the four DRZDRBI alleles by computer analysis, and 3 restriction endonucleases, FokI, Cfrl3I and HphI, were selected to detect allele-specific cleavage and RFLP patterns after PCR-amplification followed by enzyme digestion and found to produce the band patterns expected from Table 3 (Fig. 3). These 3 restriction endonucleases predict discrimination of all of 10 possible homozygous and heterozygous combinations by the DRZDRB1 alleles. D R b D R B l genotyping
Eleven subtypes of the DR4 antigen (DRB1*0401 to DRB1*0411) were determined by the modified PCR-RFLP method with 6 restriction enzymes listed in Table 4. After selective amplfication of the DR4-DRB 1 gene using the DR4-specific primers (5'R4 and 3'R). 11 subtypes can be typed by cleavage patterns with SacII, AvaII, HinfI and HaeII restriction enzymes and RFLP patterns with HphI and MnlI enzymes (26). All 66 possible combi-
nations (Tables 4, 5 and 6) of the DR4-DRB1 alleles in homozygous and heterozygous ways can be unequivocally discriminated with the restriction endonucleases selected here, except for nine combinations (DRB1*0403/0408 and 0404/0407, DRB1*0401/0411 and 0403/0409, DRB1*0404/ 041 1 and 0403/0410, DRB1*0408/0411 and 04031 0405, DRB1*0408/0411 and 0407/0410, DRBl*0403/0405 and 0407/0410, DRB1*0401/ 0410 and 040l0409, DRB1*0404/0405 and 0408/ 0410, DRB1*0401/0405 and 0408/0409) which give the identical cleavage and RFLP band patterns. As shown in Table 7, these heterozygotes, which cannot be discriminated by the PCR-SSO method theoretically (Fig. 4), can be distinguished by the use of double digestion (for example, for discrimination between SacII + HphI DRB1*0403/0408 and 0404/0407; see Fig. 4) in the PCR-RFLP method which allows examination of whether two polymorphic sites are located on the different chromosomes (trans position) or on the same chromosome (cis position). DRw52-associated (DR3, DRwl1, DRwl2, DRwl3, DRwl4 and DRw8) antigen-DRBI genotyping
Twenty-two suballeles of DR3, DRwl 1, DRwl2, DRwl3, DRwl4 and DR8 in the DRw52-associated group can be distinguished by the modified
Table a. Correlation between cleavage and RFLP patterns obtained by restriction endonucleases and DRB1 alleles for DR3,5.6 and w8 antigens Restriction Endonucleases Hphl DRBl alleles
0301 0302 1101 1103-4 1102 1201 1202 1301 1302 1303 1304 1305 1401 1404 1402 1403 1405
oaoi
0802 0804 0803
Group
Avall
Fokl
Kpnl
Haell
Cfrl31
sfaNl
Sacll
Bsall
Apal
3 3
A
1 0 0 0
1 1
C C 0
0 0 0
0 0
5u 1)
1 1 0 0 0 0 0 0 0 0 0 0 1
0
0
1 1
0 0 0 0 0 0 0 0 0
0 0
I 0
0 0 0 1 1 0
0 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 0 0 0 0 0 1 1 0 0 1
0 0 1 1 1 1 1 1 1 1 1 1
0 0 1 1 1 1 1 1
0 0 0 0 0 0 0
1
0 0
0 0
0 0 1
DR
5(11) 5U1) 5U2) 5(12) 6U 3) 6U3) 6 s 3) W3) 4 13) 6U 4) W4) 6U4) 6V4) 6U 4)
a a a a
B
E F G G H H I J J K L M N 0 0 P
0 1 1 0 1 1 1 1 0 0
1
, o
1
0 0
0 0
0
0 0 0 0 0
1 1
0 0 0 0
0
0 0 1
0
0 0 0 0 0 1 0 0 1
1 1
1 0 1
1 0
0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1
109 110
145
120
+ +
+
+ -
+ +
-
o
+ + + +
0 0 0
+ -
+ +
-
-
+
+ +
o
+ + -
0 1
0 1 1 1 1
0: not cleaved, 1: cleaved, +: positiie, -: negative.
193
Ota et al. Table 9. Cleavage patterns obtained by restriction enzymes in DRBl heterozygotes with DR3,5,6 and w8 alleles Combinations of DRB1 alleles
0301/0302 0301/110(1,3-4) 0301/I 102 0301/7201 0301/1202 0301/130(1,2) 0301/130(3,4) 0301/1305 0301/140(1,4) 0301/1402 0301/I 403 0301/1405 030110801 0301/080(2.4) 03011oao3 0302/110(1.3=4) 03031 102 03031201 03031202 0302/130(1,2) 0302/130(3,4) 03031305 0302/140(1,4) 0302/1402 0302/1403 030211405 03030801 0302/080(2.4) 0302/0803 110(1,3=4)/1102 110(1,3=4)/1201 110(1,3=4)/1202 110(1,3=4)/130(1,2) 11O( 1,3=4)/130(3,4) 110(1,3=4)/1305 110(1,3-4)/140(1,4) 110(1,3-4)/1402 110(1,3=4)11403 11O( 1,3=4)/1405 110(1,3=4)/0801 110(1,3-4)/080(2,4) 110(1,3-4)10803 1102/1201 1102/1202 1102/130(1,2) 1102/130(3,4) 1102/1305 1102/140(1.4) 1102/1402 110211403 1102/1405 110210801 1102/080(2,4) 1102/0803 1201I1 202 1201/130(1,2) 1201I130(3,4)
194
Restriction Endonucleases Groups
Avall
Fokl
Kpnl
Haefl
Crfl3l
SfaNl
Sacll
BsaJl
Apal
AiB
1 2 2 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 2 2 2 1 1 1 1 2 2 2 0 0 0 0 0 0 2 2 2 2 0 0 0 0
0 0 2 2 0 2 2 0 0 0 0 0 0 0 2 0 2 2 0 2 2 0 0 0 0 0 0 0 2 2 2 0 2 2 0 0 0 0
2 2 2
0 0 0
1 2 2
2
0 0 0 2 0 0
1 1 1 2 2
0 2 2 2 2 2 2 2
0 2 2 2 2 2 2 2 2
0 0 0 0 0 0 0 0 0
2
0
2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 0 2 2 2 0 0 0 0 0 0 0 0 0 2
A/C AID
AtE A/F
AIG AIH A/I A/J A/K AJL
AIM A/N
A/O AtP 0/c BID BIE
B/F BIG BIH 811 B/J B/K BIL B/M
BIN BIO
B/P C/D
C/E” C/F C/G*) CIH CII CIJ CIK CIL CIM” CIN CIO CIP’ D/E DIF” DIG OIH
0/l2’ DIJ DIK DIL DIM” D/N” D/O
O/P E/F
VG UH
0 0 0 0 2 2 2 2 0 0 0 0 0 0
0 0 0
2 1 2 1 1
2 2 2 2 2 2 2 1
2 1 1
2 2 2 2 2 2 2 2 2 2 2 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0
0 0
0 2 0 2 0 0 0 0 0 2 0 0 0 0 0
2 0 2 0 0 0 0 2 0 0 0 0 0 2 0 2 0 0 0 2 0 0 0 0 0 2 0 2
0 0 2
1
1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1
1 0 2 2
2 2 1 2 1 2 1 1 1 2 1 2 2 2 2 2 1 2 1 2 1 1 1 2 1
2 0 0 0 2 0 2 0 2 2 2 0 2 0 0 0
2 0
2 0 2 2 2 0 2 0 0 2 0
0
2 0 0 0 0 0 2 2 2 2 2 2 2 0 2 0 0 0 0 0 1 1
1 1 1
1 2
1 2 2 2 2 2 1
1 1 1 1 2 1 2 2 2 2 2 1 1 1
2
2 2 2 2 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
2 2 2 0 0 0
0 0 0 0 0 2 0
2 2 2 0 0 0 0 0 0 0
2 0 2 2 2 0 0 0
Modified PCR-RFLP for DRBl Table 10. RFLP patterns detected with Cfrl3I+Fokl, Fokl+SiaNI, Avall+SfaNI, Haell+ Fokl, Fokl+Apal and Sacll+Avall double digestion or Rsal digestion Cfrl31+Fokl (bp)
Combinations of
DRB1 allel6 110(1,3,4)/1201 1102/1202
DRB1 alleles
HIA-DRB1 genotyping from healthy Japanese
groups
265
201
170
CE DF
+
+
+ +
Combinations of groups
-
- Fokl+SfaNI (bp) 265
170
145
-
+
110(1,3,4)/130(1,2) 1102/1305
CG DI
i.
1201I1305 1202/130(1 ,Z)
El FG
-.
groups
265
181
145
CM IJ
+
+
+
~~
~
+
4-
+
+ -
-I-
+
Avall+SfaNI (bp)
DRB1 alleles 110(1,3,4)/1405 13051140(1,4)
__
Combinations of
Discussion
+
groups
265
170
159
110(1,3,4)/0803 1102/0801
CP ON
+
.-
+
120110801 1202/0803
EN FP
.-
+
.t
-
+ + + +
130(1,2)10801 130(3,4)/080(2,4) 1305/0803
GN
._
+-
HO
.t
IP
f
Combinations of
DRB1 alleles
groups
130(1.2)/0801 130(3,4)1080(2,4) 130510803
GN HO IP
Combinations of groups ~~~
IL KO
Combinations of
DRB1 alleles 130(3.4)/1403 140Z0803
+: positive, -: negative.
-
+ +
-
-
+
Fokl+Apal (bp)
265
205
170
+
+ + -
+ + +
-
DRBl alleles
11OZ1405 130(1,2)/140(1,4)
The HLA-DRB I genes which encode p chain molecules of DR antigens defined by the serological method, as well as Dw antigens recognized by
Haell+Fokl (bp)
DRB1 alleles
DRBl alleles
HLA-DRB 1 DNA typing was performed for genomic DNAs obtained from healthy Japanese volunteers. Generation of PCR-amplified bands with the DRB 1 group-specific primers indicated a complete agreement with DR antigens assigned serologically. Fig. 7 shows the heterozygous DRBl allelic patterns obtained by the modified PCR-RFLP method in 3 individuals (DR2/DR4; DRB1*1502/ 0401, DR4/DR6;DRBI*0405/1403, DR6/DR6; DRBI * 1303/ 1403).
~
Combinations of
130511403 1402/080(2,4)
except ones including DRBI* 1103 or 1104, as mentioned above.
-
207 ~
0 1 1 0
181
~
+ -
+
+
Rsal (bp)
150
140
111
OM GJ
+
4-
+ +
groups
111
101
HL KP
+
i.
groups
1
Sacll+Avall (bp)
265
+
1
+
81
+ -
2 2 0 2 1 1 2 2 2
Figure 7. Cleavage patterns of polymorphic restriction fragments in the PCR-amplified DRBl exon 2 genes obtained from DNAs of 3 normal individuals (No. I; DRB1*1502/0401, No. 11; DRB1*0405/ 1403, No. 111; DRBI*I303/1403) after digestion with restriction enzymes. Their genotypes were determined on the basis of Tables 3, 4, 8 and 9.
197
Ota et al. Table 11. Patterns of polymorphic fragments detected with Hphl digestion, Fokl+Hphl or Sacll+Hphl double digestion for discrimination of heterozygotes between DRB1.1101 or 1103-4 allele and the other DR3,5,6 or w8-DRBl alleles Hphl (bp) Combinations of DRB1 alleles
145
120
+ + + + + + +
+ + + + +
030111101 03011110314 03081 101 03081103-4 110111101 110111103-4 1103=4/1103=4 110211101 110211103=4 1201/1101 120111103-4 1202/1101 120211103=4 1302/1101 130111103=4 130111101 130211103-4 130311101 1303/1103-4 1304/1101 1304/1103=4
+ + + + +
* *
A A
+ * *
B B
+ +
*
.
c
c
11
-
+
+
-
+
+
+ +
-
+ +
+
-
+ + +
-
+ +
+ + + -
+
+ +
+
+
+
-
+
-
+ +
+ + +
-
-
-
+ + + + + +
+ + + + + t
-
+ + + + + + +
+ + + + +
+ + + + -
-
-
+ + + + + -
+
+
+
+
+
+
-
+
+
+
+
080311101 080311103-4 ~
+ + + + + + +
+ + +
+ + + +
+ +
35
+ + + + - + + -
+ , + + + + +
130511101 130511103- 4 140111101 140W 10314 140411101 1404/1103=4 1402/1101 1402/1103=4 1403/1101 1403/1103=4 1405/1101 1405/1103=4 080111101 080111103=4 0802/1101 0802/1103=4 0804/1101 0804/1103=4
110 109
+ + +
-
+
+
+ + +
+
+
-
+ + +
+
+
+ + +
+ +
120
+
109
+
-
95
+ -
84
+
25
+ +
11
+ +
Fokl+Hphl (bp) (*B)
145
+ +
120
+ -
109
95
+
+
-
-
84
25
11
+
-
+ +
+ +
58
47
35
+
+ +
+ +
+
Fokl+Hphl (bp) (.A)
145
-
+ -
+
+
+
Sacll+Hphl (bp) (.C)
145
+
+ -
+ +
120
+
110
+ +
62
+ +
+ -
+
+ +
11
+ +
~
+: positive, -: negative. Three pairs of *A, 08and 4 can be discriminated by RFCP band patterns obtained after double digestion with Fokl+Hphl and Sacll+ Hphl, respectively.
cellular typing, became assignable for a high degree of their polymorphisms, type at the nucleotide level using PCR procedures such as PCR-SSO typing with raido-labelled oligonucleotide probes (27-28) and with non-radioactive oligonucleotide probes (29). However, the PCR-SSO method requires multiple SSO probes, especially for the DRBI genes with as many as 43 allles, as well as labeling and washing temperatures with several stringencies 198
(27-33). A non-radioactive “reverse dot blot” method has also been used for SSO typing (15), but the amount and length of oligonucleotide probes must be adjusted and suitable conditions for hybridization determined, which is technically demanding. In this study, in order to establish a simple and practical PCR-DRB 1 genotyping, the modified PCR-RFLP method which depends mainly on cleavage or no cleavage of the amplified
Modified PCR-RFLP for DRBl Table 12. Patterns of polymorphic fragments detected with Hphl digestion, Haell+Hphl and Sacll+Hphl double digestion for discrimination of heterozygotes between DRB1.1301 or 1302 allele and the other DR3,5,6 or wBDRB1 alleles HDhl (bo) Combinations of DRB1 alleles
030111301 0301/1302 0302/1301 0302/1302 11OZ1301 11OZ1302 12011'1301 1201/1302 120Z1301 1202il302 1301/1301 1301/1302 1302/1302
145
120
110 109
35
11
+ + + +
+ + + -
+ +
-
-
+ + + + + , + + + + + + +
+ +
+
+
+
1303/1301 1303/1302 13041301 1304/1302
*
A
.
A
+
+
1305/1301 1305/1302 1401/1301 140111302 1404/1301 1404/1302 1402/1301 1402/1302 1403/1301 140311302 1 40511301 1405/1302 0801/1301 0801/1302
*B
0803/1301 0803/1302 ~~
+: positive, -: negative. Two pairs of *A
+
+
+
+ + + + + + + + + + + +
+ + + + + + + + + -
-
-
+
-
+
+ + + + + + + + + +
+ -
+ + + + + +
+ + + -
+ + +
-
+ -
-4
+
-
+ -
-
+
-
+
+
+
+ + + + +
+
+
+
+
.B
0802/1301 0802/1302 0804/1301 0804/1302
+ + -
+
-
+
+
+
+
+ + +
-
+
+
-
-
-
-
-
-
+ + + + +
+ + + +
+
~
+
+ + + + + + + + + + + + + + +
+ + +
145
120
+
-
145
120
+
+
+
+
Haell+Hphl (bp) (.A) 109 106 95
+
-
14
11
+
+
Sacll+Hphl (bp) (4) 110 62 58 47
+
+
+
-
35
11
+
+
-
+
+
+
-
-
+
+
+
-
+
+
+
+
+ ~~
~
~~~~~
and *B can be discriminated by RFLP band patterns obtained after double digestion with Haell+Hphl and Sacll+
Hphl, respectively.
segment by informative restriction enzymes was applied to the DRBl gene. This modified PCRRFLP method also utilizes allele - or group-specific primers in order to avoid cross hybridization with other DRBZ-DRBS genes and can distinguish all the 43 previously described alleles to date differing in amino acid sequence in the PI domain in homozygous and heterozygous ways, except for DRB1*1103 and 1104. The PCR-RFLP method is a simple, practical and reproducible technique for accurate definition
of HLA alleles without the use of multiple probes, and their labeling and can clearly discriminate even one base difference between alleles by restriction enzyme. Further, one of the advantages over the PCR-SSO method is that PCR-RFLP can show linkage between two polymorphic sites of interest by measurement of the length of a fragment generated after double digestion with two restriction enzymes (cis or trans position, Fig. 4), while the PCR-SSO cannot provide any such information. A characteristic feature of genetic polymorphisms 199
Ota et al. Table 13. Patterns of polymorphic fragments detected with Hphl digestion, Hphl+Sac11 double digestion for discrimination of heterozygotes between ORB1+1303or 1304 allele and the other OR 3,5.6 or w8-ORB1 alleles Hphl (bP) Combinations ot DRBl alleles
0301I1 303 0301I1304 0302/1303 030U1304 11 02/1303 11 02/1304 120111303 1201I1 304 120U1303 120U1304 1303I1303 1303/1304 130411304 1305I1303 1305/1304 1401I1 303 140111304 1404/1303 1404/1304 1402/1303 1402I1304 140311303 140311304 140511303 140511304 080111303 0801I1304
145
120
110 109
35
11
+
+
+
-
+
+
-
+
+ + +
+ +
+
-
+
+
+
+
+
-
+
+
+
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + -
+ + + + + + + + + + + + + + -
+
+
-
+ , +
-
0802/1303 0802I1304 0804/1303 0804/1304 0803/1303 0803/1304
+: positive, +: strong positive, -:
+
* *
A A
+
-
+ +
+ +
+
+ +
-
-
+ +
-
-
+ + + + +
+ + + +
-
+ + +
-
-
+ + -
+
+ +
+ +
+ + + + + + +
+
+
+
+ +
+
+ +
+ +
+
+ +
-
Sacll+Hphl (bp) CA)
145
+ +
120
+
110
+ +
62
+ +
58
+ -
47
+
35
11
+
+
+
+
+ +
negative, pair of *A can be discriminated by RFLP band patterns obtained after double digestion with Sacll+Hphl.
in the HLA genes is that they generally have no allele-specific nucleotide sequences, but that alleles differ in combinations of variable regions by taking patchwork structure. Therefore,. 15 pairs of heterozygotes* so far examined in the DRBI gene, with the same combinations of variable regions, result in the same reactive patterns to SSO probes in the PCR-SSO method as in the case of DRB1*0403/ 0408 and DRB1*0404/0407 in Fig. 4. In contrast, in the PCR-RFLP method double digestion gives *I DRB1*0401/0411 and 0403/0409, DRB1*0404/0411 and 0403/0410, DRB1*0408/0411 and 0403/0405, DRB1*0408/ 041 1 and 0407/0410, DRB1*0403/0405 and 0407/0410, DRB1*0403/0408 and 0404/0407, DRB1*0401/0410 and 040410409. DRB1*0404/0405 and 0408/0410, DRB1*0401/ 0405 and 0408/0409, DRB1*1101/1301 and 1104/1302, DRB1*0804/1101 and 0802/1104, DRB1*0892/1301 and 08041 1302, DRBl *I 103I 1201 and 1 102/ 1202, DRBl*0801 / 1102 and 0803/1103, DRB1*0801/1201 and 0803/1202.
200
-
the information on the linkage between two polymorphic sites by the appearance of the cleaved fragments which allow unequivocal discrimination between such heterozygotes. Incomplete or partial digestion of the PCR products by restriction enzymes, which will obscure definite assignment of HLA alleles can be overcome by preparation of positive control DNAs for ensuring that the digestion is complete. Further, PCR primers incorporating recognition sites for recognition enzymes used here will be more useful as an internal control. Unusual patterns of cleavage by restriction enzymes virtually indicate the presence of new alleles in the HLA genes, which must be confirmed by determination of their nucleotide sequences. This modified PCR-RFLP method could also be successfully applied to complete DRB 1, DQAl ,
Modified PCR-RFLP for DRBl Table 14. Patterns of polymorphic fragments detected with Hphl digestion, Apal +Hphl double digestion for discrimination of heterozygotes between DRBl.1401 or 1404 allele and the other DR 3,5,6 or w8-DRBl alleles Hphl (bp) Combinations of DAB1 alleles 0301/1401 0301/1404 030211401 030211404 110211401 110211404 1201/1401 120111404 120211401 120211404 130511401 1305/1404 140111401 1401/1404 140411404 1402/1401 140211404 140311401 1403/1404 140511401 140511404 0801/1401 0801I1404
145
120
110 109
+
+ +
+ + +
+
+ + + + + ' + + + + +
+ + + +
I+ . ++ +
+ +
-
+ + + + +
+ + + + + + + -
+
+ + +
+ + + + + + +
-
+ + + +
+ + + + + + + + +
35
11
-
-
+ +
-+
+ +
+ + + + + +
+ -
-
+ + -
+ + + + + + Apal+Hphl (bp) 1.44)
0802/1401 0804/1401 080211404 0804/1404 0803/1401 080311404
*A *A *B *B
+ + + -
+ + + + + +
+ + + + + +
+ + + + + +
+ + + + + +
145
+ + -
120
+ + + +
110
+ + + +
60
+ + + +
49
+ + -
35
+ + + +
11
+ + -
+: positive, +: strong positive, -: negative, pairs of *A and *B can be discriminated by RFLP band patterns obtained after double digestion with Apal+Hphl.
and DQB 1 genotyping as reported previously (22-23). This genotyping analysis can be made easily accessible to automation with a personal computer program for identification of HLA alleles. Further, PCR-RFLP is much cheaper than PCRSSO typing, especially for small numbers of samples. Thus, the PCR-RFLP method will be substituted for conventional serological and cellular typing and a good alternative to the PCR-SSO method. Acknowledgments
We thank Dr. J. Trowsdale of Imperial Cancer Research Fund for critical reading of the manuscript. This work was supported by a grant-in-aid for scientific research in 1991 from the Japanese Ministry of Education.
References 1. Bach FH. Class I1 genes and products: HLA-D region. Imrnunol Toby 1985: 6: 89-94. 2. Zinkernagel RM, Doherty PC. Restriction of in vitro T
3. 4.
5.
6. 7.
cell mediated cytotoxicity in lymphocytic choriomeningitits within a syngenic or semiallogenic system. Nature 1974: 248: 701-3. Babbit BP, Allen PM, Matsueda G, Haber E, Unanue ER. Binding of immunogenic peptides to Ia histocompatibility molecules. Nature 1985: 317: 359-61. Buus S, Sette A, Colon S, Miles C, Grey HM. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science 1987: 235: 1353-8. Guillet JG, Lai MZ, Briner TJ, et al. Immunological self/ nonself discrimination. Science 1987: 235: 865-70. Schwartz RH. T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Ann Rev Immunol 1985: 3 237-50. Bach F, Sachs DH. Transplanted immunology. N Engl J Med 1987: 317: 489-92.
201
Ota et al. Table 15.
Patterns of polymorphic fragments detected with Hphl digestion for discrimination of heterozygotes between DRB7.0802 or 0804 allele and the other DR 3,5,6 or w8-DRB1 alleles Combinations of DRB1 alleles
0301/0802 0301/0804 0302/0802 0302/0804 1102/0802 1102/0804 1201/0802 1201/0804 1202i0802 1202/0804 1305/0802 1305/0804 1402/0802 1402/0804 1403/0802 1403/0804 1405/0802 1405/0804 0801/0802 0801/0804 0802/0802 oao2/oao4 0804/0804 0803/0802 0803/0804
Hpbl (bp)
145
120
110 109
35
11
+ +
+
+ + + + + +
+ -
+ i.
+ + + + +
-
-I.
+ -
+
+ + -
+
+ + +
+ + +
+ + + ' + + + + + + + + + + + +
+
+ +
+
+ + +
+ + + + + + + + + +
+
+ + + + + +
+ +
+
-
+
+ -
+
+ + + + + + -
+
+ + + + +
+: positive, -: negative. 8. Khagani A, Yacoub M, McCloskey D, et al. The influence of HLA matching, donor, recipient sex, and influence of acute rejection on survival in cardiac allograft ren'pients receiving cyclosporin A and Azathioprine. Transplant Proc 1989: 21: 799-800. 9. Todd JA, Acha-Orbea H, Bell JI, et al. A molecular basis for MHC class 11-associated autoimmunity. Science 1988: 240: 1003-9. 10. Marsh SGE, Bodmer JG. HLA class I1 nucleotide sequences. Tissue Antigens 1991: 37: 181-9. 1I . Marsh SGE, Bodmer JG. DR and DQ epitopes and monoclonal antibody specificity. Immunol Today 1990: 1 0 305-1 I. 12. Grosse-Wilde H, Doxiadis I, Brandt H. Definition of HLAD with HTC. In: Albert MT, Baur MT, Mayr WR, eds. Histocompatibility Testing 1984. Heidelberg: Springer-Verlag, 1984: 249-64. 13. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of P-globin genominc sequence and restriction site analysis for diagnosis of sickle cell anemia. Science 1985: 230: 1350-4. 14. Mullis KB, Fallona F. Specific synthesis of DNA in vitro via polymerase catalyzed chain reaction. Methodr Enzymol 1987: 155: 335-50. 15. Saiki R, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequencespecific oligonucleotide probes. Proc Natl Acad Sci USA 1989: 86: 6230-4. 16. Trucco G, Fritsch R, Giorda R, Trucco M. Rapid detection of IDDM susceptibility with HLA-DQ-alleles as markers. Diabetes 1989: 38: 1617-22.
17. Yunis I, Salazar M, Yunis EY. HLA-DR generic typing by AFLP. Tissue Antigens 1991: 38: 78-88. 18. Maeda M, Murayama H, Ishi H, et al. A simple and rapid method for HLA-DQAl genotyping by digestion of PCRamplified DNA with allele specific restriction endonucleases. Tissue Antigens 1989: 3 4 290-8. 19. Maeda M, Uryu N, Murayama H, et al. A simple and rapid method for HLA-DP genotyping by digestion of PCRamplified DNA with allele-specific restriction endonucleases. Human Immunol 1990: 27: 111-21. 20. 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 specific restriction endonucleases. Tissue Antigens 1990: 3 5 20-31. 21. Olerup 0. HLA-class I1 typing by digestion of PCR-amplified DNA with allele-specificrestriction endonucleaseswit1 fail to unequivocally identify the genotypes of many homozygous and heterozygous individuals. Tissue Antigens 1990: 36 83-7. 22. Nomura N, Ota M, Tsuji K, Inoko H. HLA-DQBI genotyping by a modified PCR-RFLP method combined with group-specific primers. Tissue Antigens 1991: 38: 53-9. 23. Ota M, Seki T,Nomura N, et al. Modified PCR-RFLP method for HLA-DPB1 and -DQAI genotyping. Tissue Antigens 1991: 38: 60-71. 24. 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. 25. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymaticamplification of DNA with a thermostable DNA polymerase. Science 1988: 239: 487-9 I. 26. Ota M, Seki T, Kiyosawa K, et al. A possible association between basic amino acids of position 13 of DRBl chains and autoimmune hepatitis. Immunogenefics 1992 (in press). 27. Vaugham RW, Lanchburg JSS, Marsh SGE, Hall MA, Bodmer JG, Welsh KI. The application of oligonucleotide probes to HLA class I1 typing of the DRB subregion. Tissue Antigens 1990: 36: 149-55. 28. Mach B, Tiercy J-M. Genotypic typing of HLA class 11: from the bench to the bedside. Human Immunol 1991: 30: 278-84. 29. Scharf SJ, Griffith RL, Erlich HA. Rapid typing of DNA sequence polymorphism at the HLA-DRBI locus using the polymerase chain reaction and nonradioactive oligonucleotide Drobes. Human Immunol 1991: 30: 190-20 1. 30. Gao X,.Moraes JR, Miller S, Stastny P. DNA typing for class I1 HLA antigens with allele-specific or group-specific amplification V. Typing for subsets of HLA-DR1 and DR'Br'. Human Immunol 1991: 30: 147-54. 31. Moraes ME, Vina MF, Stastny P. DNA typing for class I1 antigens with allele-specificor group-specificamplification. IV. Typing for alleles of the HLA-DR2 group. Human Immunol 1991: 31: 139-41. 32. Gao X, Vina MF, Shumway W, Stastny P. DNA typing for class I1 HLA antigens with allele-specific or group-specific amplification. 1. Typing for subsets of HLA-DR4. Human Immunol 1990: 2 7 40-50. 33. Petersdorf EW, Smith A, Mickelson EM, Martin PJ, Hansen JA. Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRBl first domain. Immunogenefics 1991: 33: 26.5-71. 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: 0263-34-8480
