Molecular Analysis of HLA-DRB 1"08/12 Alleles: Identification of Two Additional Alleles* Mary Eberle and Lee Ann Baxter-Lowe
ABSTRACT: A nonradioactive oligotyping method that takes advantage of selective amplification using the polymerase chain reaction (PCR) and oligonucleotide probe hybridization was developed to distinguish all reported HLA-DRB 1"08/12 alleles. Selective amplification was achieved using a primer complementary to the sequence encoding YSTGECY at positions 10-16 in the first hyperpolymorphic region (HPMR). This selective amplification of the HLA-DRBl*08/12 subset of alleles provides a refinement in HLA oligotyping that permits unambiguous oligotyping of many heterozygotes that cannot be resolved using less selective amplification alternatives. The amplified DNA was hybridized with a panel of then digoxigenin-labeled probes to resolve oligotypes
ABBREVIATIONS PCR polymerase chain reaction
that correspond to all reported HLA-DRBl*08/12 alleles. Oligotyping of HLA-DRB1*08/12 samples revealed two previously unknown HLA-DRB alleles. One allele, DRBI*0805, differs from DRBI*0801 by a leucine to alanine substitution at position 74. This allele is of particular interest because it is very similar to HLADRBI*08 alleles (YSTGECY and lack of an associated HLA-DRB3 gene), but it lacks leucine at position 74, which is characteristic of all previously reported DRBI*08 alleles. The second HLA-DRBI*08 allele, DRBI*0804, differs from DRBI*0802 by a glycine to valine substitution at position 86. Human Immunology 34, 24-30 (1992)
HPMR
hyperpolymorphic region
INTRODUCTION H L A oligotyping, a method that utilizes sequence-specific oligonucleotide hybridization to detect polymorphic sequences [ 1 - 3 ] in amplified D N A , provides a practical means of routine H L A typing. Advantages of
* T h e names DRBI*0804 and DRBI*0805 were officially assigned to these new sequences by the W H O Nomenclature Committee in N o v e m b e r 1991. This follows the agreed policy that, subject to the condinons stated in the most recent Nomenclature Report [10], names will be assigned to new sequences as they are identified. Dsts of such new names will be published in the forthcoming W H O Nomenclature Report. The DRBI*0804 and DRBI*0805 sequences have been submitted to Gen Bank and assigned the accession numbers M844446 and M84357, respectively.
From the Blood Center of Southeastern Wisconsin and the Medical College of Wl~consin, Milu,aukee, Wisconsin. Address reprint requests to Dr. Lee Ann Baxter-Lou,e, The Blood Center of Southeastern Wisconsin, 1701 West Wisconszn Avenue, Milwaukee, WI 5.3233. Received August 15, 1991: aaeptedJanuary 2, 1992. 24 0198-8859/92/,$5.00
oligotyping include the ability to type a wide range of samples (e.g., nonviable cells and cells with low expression of H L A proteins) and the use of synthetic rather than biologic reagents. Another advantage of oligotyping is that specific amplification can reduce or eliminate ambiguity resulting from the sharing of a single polymorphic sequence by multiple alleles. This is accomplished by controlling the specificity of each amplification to generate amplified products of one locus, a particular subset of alleles, or an individual allele {4-7]. Analysis of a single allele or subset of alleles is essential for unambiguous assignment of oligotypes in certain heterozygous individuals. A method to oligotype all H L A - D R B 1 alleles that are associated with H L A - D R w 5 2 haplotypes has been reported [7]. H o w e v e r , due to the sharing of polymorphic sequences within this group, many heterozygous combinations cannot be resolved. This limitation was Human Immunology 34, 24-30 (1992) © American Society for Histocompatibility and lmmunogenetics, 1992
HLA-DRB l'D08/12 Alleles
25
TABLE 1 Primers utilized for HLA-DRB1*08/12 oligotyping Name
Sequence
Positive hybridization
PCR6 PCR5 PCR44 T7 M13forward
5'-ATTTCTTCAATGGGACGGAGC-3' 5'-CGCCGCTGCACTGTGAAGCTCTC-3' 5 '-GTACTCTACGGGTGAGTGTT-3' 5 '-AATACGACTCACTATAG-3 ' 5 '-GTTTTCCCAGTCACGACGT-3'
All DRB alleles All DRB alleles DRB 1*08, 12, 1404 pCR1000 vector pCR1000 vector
circumvented by developing an oligotyping method that utilizes a subset of HLA-DRw52 associated alleles containing YSTGECY (HLA-DRB 1*08/12/1404). Amplified DNA was hybridized to a panel of probes that differentiate all oligotypes associated with this subset of alleles. Using this method, two additional HLADRBI*08 alleles were detected and were confirmed by direct sequencing.
MATERIALS A N D M E T H O D S
Oligonucleotides.
Oligonucleotides were synthesized using either a Gene Assembler (Pharmacia Fine Chemicals, Piscataway, NJ) or a polymerase chain reaction (PCR) Mate (Applied Biosystems, Foster City, CA). The sequences of all primers and probes used in this study are provided in Tables 1 and 2. Probes were named as the polymorphic amino acid(s) encoded by the segment of the gene complementary to the oligonucleotide and a number corresponding to the position of the polymorphic amino acid(s) in the sequence. For example, L37L38 detects the nucleotides encoding two leucine residues found at positions 37 and 38 in DRBl*1201 and 1202.
TABLE 2
DRB codons 16-22 87-94 10-16
Oligotyping. Genomic DNA was isolated as described previously [8] from B-lymphoblastoid cell lines or white blood cells that were typed as HLA-DRw8 and -DRwI2 using serologic methods. Amplification was achieved using the PCR as described earlier [9] with the modifications described below. Intralocus-specific amplification utilized a 5' primer (PCR44) complementary to sequences encoding YSTGECY at positions 10-16 and a 3' primer (PCR5) complementary to codons 8794 of all HLA-DRB alleles (Table 1); MgCl2 was 3.5 mM; and thermal cycling was 20 seconds at 98°C, 30 seconds at 55°C, and 30 seconds at 72°C. Locus-specific amplification utilized a 5' primer (PCR6) that is complementary to most nucleotides at codons 16-22 of all HLA-DRB alleles and PCR5 (Table 1); MgC12 was 1.5 mM; and thermocycling was 15 seconds at 98°C, 20 seconds at 55°C, and 15 seconds at 72°C. Amplified DNA was hybridized with digoxigenin-labeled oligonucleotide probes as described earlier [9] using temperatures for probe hybridizations and washes that are listed in Table 2. The conditions necessary for obtaining the appropriate specificities of primers and probes were determined by oligotyping the B-lymphoblastoid cell lines that were characterized during the Tenth International Histocompatibility Workshop.
Oligonucleotide probes utilized for HLA-DRB1*08/12 oligotyping
Name
Codons
Sequence
L37L38 F37 $57 con6 la I67 F67 A74 L74 G86 V86
36-39 36-39 56-59 61-64 67-71 67-71 70-74 71-74 85-88 85-88
5'-CGCAGGAGCTCC-3' 5'-CGCACGAACTCC-3' 5'-CTCGGCGCTAGG-3' 5 '-TCTGGCTGTTCC-3' 5'-CCTGTCTTCCAGGATG-3' 5'-CTGTCTTCCAGGAAG-3' 5'-CGCGGCCCGCCTGT-3' 5'-CAGGGCCCGCCT-3' 5'-CTCTCACCAACC-3' 5'-CTCTCCACAACC-3'
con61 is the consensus probe.
Positive hybridization "1201, 1202 * 1404 "0801, 0803, All DRB 1 *0803, 1201 "0801, 0802, *0805 "0801, 0802, "0801, 0802, "1404, 0804
0805
0804, 0805, 1202 0803, 0804 0803, 0805
Hybridization temp (°C)
Wash temp
30 35 30 30 35 35 30 30 30 30
32 35 30 30 55 45 48 32 32 32
(°c)
26
M. Eberle and L. A. Baxter-Lowe
6
10
20
30
40
60
70
80
90
RFLE" - TSECHFFMP,TERVRFLDRYFYMQEEYVRFDSOVGEYRAVTELGRPOAEYt,AISGi(DLLEDRRAAVI)TYCRHIIYGWESFiVQRR ( .... vs-6--, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s . . . . . . . . . F . . . . . . . . . . . . . . . . . . s ........ ) c .... vs-~--v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s ......... F ...... , . . . . . . . . . . . ~. . . . . . . . . ) ( .... vs-~--Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F ...... L . . . . . . . . . . . ~ ........ ) ( .... ,S-6--, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *: . . . . . . , . . . . . . . . . . . . . . . . . . . . ) ( .... YS'G-'Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S ......... I ...... L ........... G ........ ) c .... v s - ~ - - v . . . . . . . . . . . . . . . . .... F . . . . . . . . . . . . . . . . . . . A--. ......... R---E . . . . . . . . . . . . . . . . . . . . ) ( .... YS-G- -Y ......... Y-E-H-H .... LL ........ F ......... V- -S ...... l ................. A ......... ) ( .... YS-G--Y ......... Y-E-H-H .... LL ........ F ......... V--S ...... F ................. A ......... )
colrisensus
DRBI*0805 DRBI*0801 DRBI*0802 DItBI*0804 DRBI*0803 DRB1*1404 DRB1*1201 DRB1*1202
F I G U R E 1 Alignmen~ of HLA-DRB1*08/12/1404 alleles. The amino acid sequences of DRB1 alleles detected in this assay (reviewed in Marsh and Bodmer [23]) are listed with the HLA-DRB consensus sequence at the top. Dashes indicate residues that are identical to the consensus sequence and the single-letter amino acid code shows deviation from the consensus sequence.
Products from alleles e n c o d i n g Y S T G E C Y (HLAD R B 1 * 0 8 / 1 2 ) were h y b r i d i z e d with a panel o f probes (Table 2) that detect p o l y m o r p h i c s e q u e n c e s that differentiate all r e p o r t e d alleles of this g r o u p (Fig. 1 and Table 3). I n t e r n a l controls, which are used to m o n i t o r the specificity o f each hybridization, were amplified using p r i m e r s P C R 6 and P C R 5 , which are specific for H L A D R B (Table 1). A c o n s e n s u s p r o b e (Table 2), which hybridizes to all H L A - D R B alleles, was i n c l u d e d to m o n i t o r the a m o u n t o f D N A b o u n d to the m e m b r a n e . N o m e n c l a t u r e for oligotypes is based u p o n the designations for H L A alleles that have b e e n r e p o r t e d by the W H O N o m e n c l a t u r e C o m m i t t e e for Factors of the H L A System [10]. O l i g o t y p e s are distinguished from alleles by s u b s t i t u t i n g a dash (-) for the asterisk (*).
Nucleotide sequencing of alleles. T h e D R B 1"08/12/1404specific 5' p r i m e r ( P C R 4 4 ) was utilized to amplify the
TABLE 3
50
second e x o n (codons 1 6 - 9 4 ) o f the H L A - D R alleles selectively. Amplified D N A was ligated into the p C R 1 0 0 0 vector ( I n v i t r o g e n C o r p o r a t i o n , San Diego, CA). Plasmid D N A was isolated from clear colonies and s e q u e n c e s of the inserts from m u l t i p l e clones were det e r m i n e d using vector p r i m e r s and standard dideoxy t e r m i n a t i o n m e t h o d s [11].
Serologic HLA typing. H L A serologic typing using microcytotoxicity assays [12] was g e n e r o u s l y p r o v i d e d by the H i s t o c o m p a t i b i l i t y and I m m u n o g e n e t i c s Laboratory o f the B l o o d C e n t e r of S o u t h e a s t e r n Wisconsin. T h e serologic specificities for samples c o n t a i n i n g the alleles designated D R B I * 0 8 0 5 and D R B I * 0 8 0 4 were A 2 , - ; B27,44(4); Cw 1 ,w5; D R 4 , w 8 , w 5 2 , w 5 3 ; D Q w 4 , w 7 , and A30,33; B35,w42(6); Cw4,-; DR4,w8,w52,w53; D Q w 7 , w 8 , respectively.
RESULTS AND DISCUSSION
HLA-DRBI*08/12/1404 oligotyping. A nonradioactive oligotyping system was d e v e l o p e d to p r o v i d e a practical, c o m p l e t e system o f typing H L A - D R B 1 * 0 8 / 1 2 / 1 4 0 4 alleles. This subset of H L A - D R B 1 alleles was specifically amplified to r e d u c e the complexity of data interpreta-
H y b r i d i z a t i o n p a t t e r n s o b t a i n e d for H L A - D R B 1"08/12 oligotyping Oligotype
Probe
0805
L37L38 F37 $57 con61 167 F67 A74 L74 G86 V86
. . ÷ + . + + + -
0801 . .
0802 .
.
.
+
+
+ .
+ + -
1201
1202
+ + -
÷ + + -
÷ + +
-
-
.
. .
1404
.
+ .
+ . + + -
0803
.
.
÷ + .
0804
. ÷ +
+ + + . + + -
.
. +
-
HLA-DRB 1"D08/12 Alleles
27
Probe: L74
Probe: con61 A 1
J
B
C
A
D
B
C
D
ifiiqii~
;i~ii~ii~ ....
iiiii
7
...... i;i~ii!
....
7 r;;iiiiii
9
~,,~®, i!iil; i!!!ii i,
9
lO
.............. iii!i~ !i!,;i!!;~:: ..... :ii)!ii
lO
11
i~
11
.... iiiii:i;/
FIGURE 2 Primary data from DRBl*08/12 oligotyping. A digoxigenin-labeled probe, con61, is a consensus probe that is utilized to monitor loading of DNA on membranes. Probe L74 detects the polymorphism (CTG) at codon 74. (A) Control DNA was amplified from the appropriate Tenth International Histocompatibility Workshop B-Lymphoblastoid Cell Lines and are included to monitor the specificity of hybridization. Primers (PCR6 and 5) were used to amplify all HLADRB alleles. (B-D) DNA from HLA-DRBl*08/12/1404 alleles was specifically amplified utilizing PCR44 and 5. Oligotypes were assigned as described in Materials and Methods. B7 was assigned an DRB1-0805 oligotype and thus lacks hybridization to L74. D6 is an ink dot used for orientation of membrane. Sample descriptions are provided in Table 5.
tion that results from sharing of certain polymorphic residues by multiple alleles. Selective amplification used a primer complementary to the nucleotides encoding Y S T G E C Y at positions 1 0 - 1 6 of this subset of alleles. Hybridization of the amplified D N A to a panel of ten nonradioactive probes permitted assignment of a unique oligotype to each reported allele in this group (Fig. 1): H L A - D R B I * 0 8 0 1 [13], 0802 [14, 15], 0803 [14], 0804 [7, 16], 1201 [17], and 1202 [18]. An additional allele, H L A - D R B l * 1 4 0 4 [19, 20] (assigned on "HLA-DRw8-iike" serologic specificity, classified as a member of the H L A - D R B I * 1 4 group) was also amplified using this primer and was detected in this assay. For the H L A - D R B l - 0 8 / 1 2 / 1 4 0 4 alleles, oligotypes are assigned based upon the specific pattern of hybridization (Table 3) with ten probes that detect the polymorphic sequences that distinguish these alleles from each other. For example, DRB1-0801 oligotype is as-
signed if the amplified D N A hybridizes with $57, F67; L74, and G86, and not to the remaining probes (i.e., L37L38, F37, I67, A74, and V86). Likewise, DRB11201 oligotype is assigned if hybridization was positive for L37L38 and I67 and negative for F37, $57, F67, A74, L74, G86, and V86. Figure 2 shows an example of two membranes (one with the consensus probe, con61, and one with L74) from a typical oligotyping assay. Assignment of precise oligotypes for many heterozygous individuals can be difficult due to the sharing of polymorphic residues among alleles. The probes used in oligotyping of the HLA-DRB1*08/12/1404 alleles can hybridize with many alleles, making specific amplification essential. When specific amplification of YSTGECY containing alleles is used, unequivocal assignment of oligotypes is possible for most combinations of alleles. The four combinations that cannot be differentiated are listed below: HLA-DRB 1"0801/0801 or - DRB 1"0801/0802 HLA-DRB 1"0805/0801 or - D R B 1"0805/0802 HLA-DRB 1"0801/0803 or - DRB 1"0802/0803 HLA-DRB1*0801/1201 or - D R B 1 * 0 8 0 3 / 1 2 0 2 This circumstance is uncommon because these alleles are present at a fairly low frequency [21]. A recent publication [7] describes oligotyping of alleles associated with the HLA-DRw52 supertypic group (DRB1*03/11/12/13/14/08) utilizing a primer that amplifies all alleles that encode EYST at positions 9-12. In this circumstance, at least 27 alleles are coamplified and the number of heterozygotes that cannot be resolved increases substantially. The oligotyping method described here provides a reliable, accurate method for oligotyping samples containing HLA-DRBl*08/12/ 1404 alleles and has been used to type about 100 samples successfully (Table 4).
TABLE 4
N u m b e r of H L A - D R B 1-08/12/1404 oligotypes
HLA-DRB1 oligotype
Number of oligotypes
0801 0802 0803 0804 0805 1201 1202 1404
32 14 14 1 2 35 2 0
28
TABLE 5
M. Eberle and L. A. Baxter-Lowe
Samples s h o w n in Fig. 2 Oligotype'
Position* A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Bll C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Cll D 1 D2 D3 D4 D5
Sample nameb 9010 9087 9034 9037 9057 9097 9096 9069 9075 CS 9038 M7 M10 Mll M12 M13 M14 M16 M20 M21 M22 M23 M28 M29 M31 M32 M33 M34 M39 M44 M48 M52 M54 M56 M57 M58 M59 M60
AMAI STEINLIN SAVC SWEIG TEM EMJ LBF MADURa DKB BM16
DRB11501 0301 0401 1101 1401 1302 0702 0801 0901 0701, 1001 1202 07, 12 04, 12 04, 12 15/16, 08 07, 12 01, 12 04, 08 14, 08 07, 08 04, 08 08, 11 01, 08 07, 08 03, 12 03, 08 03, 08 07, 08 03, 08 03, 08 07, 08 08, 11 08, 10 15/16, 08 08, 11 15/16, 08 08, 12 03, 08
DRB3-
DRB4-
DRB50101
0101 0101 0202 0201 0301 0101 0101 0101 0202 X
X
X X
X X
X X
X
X
X X X X X X X X X X X X X X X X X X X
Position of samples shown in Fig. 2. bSamples with four digit number preceding name are from the Tenth International Histocompatibility Workshop. Those preceded with an M are samples of indicated primary oligotypes. cOligotypes were determined to identify groups of alleles using an HLA-DR oligotyping method that approximates serologic specificities [29]. An X represents the detection of a polymorphic sequence indicative of the locus.
Discovery of novel HLA-DRB1 alleles. H y b r i d i z a t i o n patterns p r e s e n t e d in T a b l e 3 d e m o n s t r a t e the d e t e c t i o n o f u n k n o w n H L A - D R alleles using oligotyping. T h e s e alleles are revealed by the p r e s e n c e o f c o m b i n a t i o n o f p o l y m o r p h i c s e q u e n c e s that are n o t a t t r i b u t a b l e to any p r e v i o u s l y r e p o r t e d alleles. S e q u e n c e analysis o f the exon e n c o d i n g the first d o m a i n c o n f i r m e d the seq u e n c e s p r e d i c t e d by o l i g o t y p i n g (Fig. 1). T h e first allele is identical to D R B I * 0 8 0 1 except that
c o d o n 74 (Fig. 1) is G C G (alanine) rather than C T G (leucine). This s u b s t i t u t i o n is particularly i n t e r e s t i n g because l e u c i n e at p o s i t i o n 74 is characteristic o f H L A D R w 8 m o l e c u l e s and could be i n v o l v e d in the formation o f an e p i t o p e r e c o g n i z e d by H L A - D R w 8 - specific alloantisera. T h e only r e p o r t e d e x c e p t i o n is H L A D R B 1 * 1 4 0 3 , which e n c o d e s l e u c i n e at 74, but was rep o r t e d as a b l a n k by serologic typing [22]. T h e new allele H L A - D R B I * 0 8 0 5 lacks l e u c i n e at position 74,
HLA-DRB 1"D08/12 Alleles
but was serologically typed as H L A - D R w 8 . It does share two nonexclusive characteristics of other D R B I * 0 8 alleles: the lack of an associated H L A - D R B 3 allele and the presence of first H P M R sequences, Y S T G E C Y . It is difficult to understand the relationship between these two characteristics and the structural basis for the H L A - D R w 8 serologic specificity. The second allele H L A - D R B I * 0 8 0 4 differs from H L A - D R B I * 0 8 0 2 by a single amino acid substitution at codon 86, a G G T (glycine) to ( G T G ) valine (Fig. 1). This allele was also independently observed by others [7, 16]. This dimorphism is c o m m o n and has already been reported for several other H L A - D R B alleles, such as H L A - D R B l * 1 5 0 1 and 1502; D R B I * 0 4 0 4 and 0408; and DRB3*0201 and 0202 (reviewed by Marsh and B o d m e r [23]). It has been suggested that this position is important for T-cell recognition. For example, Lang et al. [24] suggest that position 86 is involved in formation of the H L A - D w l 3 and -Dw14 determinant recognized by a T-cell clone (E38). Several investigators [ 2 5 - 2 8 ] have implicated positions 86 (along with position 85) in forming H L A - D R 1 associated allodeterminants detected in cellular assays. The physiologic significance of this dimorphism at position 86 can now be tested using oligotyping systems such as that reported here in which each allele is independently examined.
Origin of the HLA-DR alleles. Each of the two alleles reported here differ from reported alleles by two-basepair differences (CT and G C at nucleic acids 2 0 7 - 2 0 8 of H L A - D R B I * 0 8 0 1 and G T to T G at 2 4 4 - 2 4 5 of HLA-DRB1*0802). G e n e conversion events provide one explanation for the origin of the new H L A - D R variants. The lack of an H L A - D R B 3 product in all H L A - D R B I * 0 8 alleles is consistent with derivation of each of these alleles from a c o m m o n ancestor with gene conversion events introducing polymorphic sequences.
Future of oligotyping classification. As additional H L A alleles continue to be discovered, it is becoming apparent that the classification of H L A alleles according to serologic specificities will become increasingly difficult. For example, the first three HLA-DRw8-associated alleles that were discovered could be distinguished from all other H L A - D R alleles by the presence of leucine at position 74, the lack of a linked H L A - D R B 3 product, and the presence of the sequence, Y S T G E C Y , in the first H P M R ( Y S T G E C Y is also found in D R B I * 1 2 alleles). At that time, it appeared that leucine at position 74 could provide the basis for assignment of an HLAD R B 1-08 oligotype and could be implicated in the formation of an HLA-DRw8-specific epitope. However, the discovery of additional alleles raises questions regarding these possibilities. For example, the HLA-
29
D R B I * 0 8 0 5 allele lacks leucine at position 74, but shares the two other distinguishing characteristics of HLA-DRw8-associated alleles. Another allele that was reported as "HLA-DRw8-1ike," D R B 1" 1404 [ 19], has alanine at position 74, but differs from H L A - D R B 1"08 alleles by linkage with an H L A - D R B 3 product. This suggests that leucine at position 74 is not essential for assignment of H L A - D R w 8 . Perhaps the D R B I * 0 8 classification should be reserved for alleles that are not linked to an H L A - D R B 3 locus and that encode Y S T G E C Y in the first H P M R or H L A - D R B 1. This classification is appealing from a teleologic standpoint, but sharing of polymorphic sequences and heterozygosity would make it difficult to detect an H L A - D R B 1-08 oligotype when products of a locus-specific amplification are examined. H L A oligotyping, a method that utilizes sequencespecific oligonucleotide hybridization to detect specific sequences in amplified D N A , is a practical method for routine H L A typing that provides a level of resolution that surpasses serologic typing. This level of resolution may be very important for selection of donors for bone marrow transplantation and studies involving the role of H L A in autoimmune disease. ACKNOWLEDGMENTS
We thank Dr. Jack Gorski for a critical review of the manuscript and insightful discussion. We are grateful to Ufuk Asu and the members of the Histocompatibility and Immunogenetics Laboratory and the DNA Diagnostics Laboratory of the Blood Center of Southeastern Wiscdnsin for providing assistance in this investigation. This study was supported by the National Institutes of Health (R01 AI28034) and the Blood Center of Southeastern Wisconsin.
REFERENCES 1. Angelini G, de Preval C, Gorski J, Mach B: High-resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 83:4489, 1986. 2. Conner BJ, Reyes AA, Morin C, Itakura K, Teplitz RL, Wallace RB: Detection of sickle cell/3S-globin allele by hybridization with synthetic oligonucleotides. Proc Natl Acad Sci USA 80:278, 1983. 3. Baxter-Lowe LA, Hunter JB, Casper JT, Gorski J: HLA gene amplification and hybridization analysis of polymorphism: HLA matching for bone marrow transplantation of a patient with HLA deficient severe combined immunodeficiency syndrome. J Clin Invest 84:613, 1989. 4. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487, 1988. 5. Gao X, Fernandez-Vina M, Shumway W, Stasmy P: DNA typing for class II HLA antigens with allele-specific
30
M. Eberle and L. A. Baxter-Lowe
or group-specific amplification. I. Typing for subsets of HLA-DR4. Hum Immunol 27:40, 1990. 6. Lanchbury JSS, Hall MA, Welsh KI, Panayi GS: Sequence analysis of HLA-DR4B 1 subtypes: additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing. Hum Immunol 27:136, 1990. 7. Fernandez-Vina M, Shumway W, Stasmy P: DNA typing for class II HLA antigens with allele-specific or groupspecific amplification: typing for alleles for the DRw52associated group. Hum Immunol 28:51, 1990. 8. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988. 9. Molkentin J, Gorski J, Baxter-Lowe LA: Detection of 14 HLA-DQB1 alleles by oligotyping. Hum Immunol 31:114, 1991. 10. Bodmer JG, Marsh SGE, Albert ED, Bodmer WF, Dupont B, Erlich HA, Mach B, Mayr WR, Parham, P, Sasazuki T, Schreuder GMTh, Strominger JL, Svejgaard A, Terasaki PI: Nomenclature for factors of the HLA system, 1991. Tissue Antigens 39:161, 1992. 11. Sanger F, Nicklen S, Coulson A: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977. 12. van Rood JJ, Van Leeuwen A, Keuning JJ, Blusse van Oud Alblas A: The serological recognition of the human MLC determinants using a modified cytotoxicity technique. Tissue Antigens 5:73, 1975. 13. Bell JI, Denney D Jr, Foster L, Belt T, Todd JA, McDevitt HO: Allelic variation in the DR subregion of the human major histocompatibility complex. Proc Natl Acad Sci USA 84:6234, 1987. 14. Gorski J: The HLA-DRw8 lineage was generated by a deletion in the DR B region followed by first domain diversification. J Immunol 142:4041, 1989. 15. Jonsson A-K, Andersson L, Rask L: A cellular and functional split in the DRw8 haplotype is due to a single amino acid replacement (DR~3Ser57-Asp57) Immunogenetics 29:308, 1989. 16. Hurley CK, Lee KW, Mickelson E, Masewicz S, Johnson AH: DRw8 microvariation: a new DRB1 allele identified in association with DQw7 in American blacks. Hum Immunol 31:109, 1991. 17. Navarrete C, Seki T, Miranda A, Winchester R, Gregersen PK: DNA sequence analysis of the HLA-DRwl2 allele. Hum Immunol 25:51, 1989. 18. Abe A, Ito I, Ohkubo M, Kaneko T, Ito K, Kato H, Kashiwagi N, Obata F: Two distinct subtypes of the
HLA-DRw12 haplotypes in the Japanese population detected by nucleotide sequence analysis and oIigonucleotide genotyping. Immunogenetics 30:422, 1989. 19. McClure GR, Ruberti G, Fathman CG, Erlich HA, Begovich AB: DRBI*LY10: a new DRB1 allele and its haplotypic association. Immunogenetics 32:214, 1990. 20. Gorski J, Radka SF, Masewicz S, Mickelson EM: Mapping of distinct serologic and T cell recognition epitopes on an HLA-DR/3-chain. J Immunol 145:2020, 1990. 21. Baur MP, Neugebauer M, Albert ED: Reference tables of two-locus haplotype frequencies for all MHC marker loci. In Albert ED, Baur MP, Mayr WR (eds): Histocompatibility Testing 1984. New York, Springer-Verlag, 1984, p 677. 22. Obata F, Abe A, Ohkubo M, Ito I, Kaneko T, Otani F, Watanabe K, Kashiwagi N: Sequence analysis and oligonucleotide genotyping of HLA-DR"JX6", a DR "blank" haplotype found in the Japanese population. Hum Immunol 27:269, 1990. 23. Marsh SGE, Bodmer JG: HLA class II nucleotide sequences, 1991. Hum Immunol 31:207, 1991. 24. Lang B, Navarrete C, LoGalbo PR, Nepom GT, Silver J, Winchester RJ, Gregersen PK: Further DNA sequence microheterogeneity of the HLA-DR4/Dwl3 haplotype group: importance of amino acid position 86 of the DR/31 chain for T-cell recognition. Hum Immunol 27:378, 1990. 25. Eckels D, Sell T, Eiermann T, Nikaein A: DR1: antigen report of the cellular studies of; the Tenth International Histocompatibility Workshop. In Dupont B (ed): Immunobiology of HLA, vol 1: Histocompatibility Testing 1987. New York, Springer-Verlag, 1989, p 502. 26. Ziff BL, Hurley CK, Rosen-Bronson S, Tang T, Hartzman R, Silver J, Gregersen P, Eckels D, Johnson AH: Polymorphism of the DR1 haplotype: structural and functional analysis. In Dupont B (ed): Immunobiology of HLA, vol 2: Immunogenetics and Histocompatibility. New York, Springer-Verlag, 1989, p 187. 27. Hurley CK, Ziff BL, Silver J, Gregersen PK, Hartzman R, Johnson AH: Polymorphism of the HLA-DR1 haplotype in American blacks: identification of a DR1/3-chain determinant recognized in the mixed lymphocyte reaction. J Immunol 140:4019, 1988. 28. Eckels DD, Geiger MJ, Sell TW, Gorski J: Involvement of class II ~-chain amino acid residues 85 and 86 in T-cell allorecognition. Hum Immunol 27:240, 1990. 29. Savage D, Baxter-Lowe LA, Gorski J, Middleton D: Molecular methods. In: Histocompatibility Testing: A Practical Approach. IRL (in press).
ABSTRACT: A nonradioactive oligotyping method that takes advantage of selective amplification using the polymerase chain reaction (PCR) and oligonucleotide probe hybridization was developed to distinguish all reported HLA-DRB 1"08/12 alleles. Selective amplification was achieved using a primer complementary to the sequence encoding YSTGECY at positions 10-16 in the first hyperpolymorphic region (HPMR). This selective amplification of the HLA-DRBl*08/12 subset of alleles provides a refinement in HLA oligotyping that permits unambiguous oligotyping of many heterozygotes that cannot be resolved using less selective amplification alternatives. The amplified DNA was hybridized with a panel of then digoxigenin-labeled probes to resolve oligotypes
ABBREVIATIONS PCR polymerase chain reaction
that correspond to all reported HLA-DRBl*08/12 alleles. Oligotyping of HLA-DRB1*08/12 samples revealed two previously unknown HLA-DRB alleles. One allele, DRBI*0805, differs from DRBI*0801 by a leucine to alanine substitution at position 74. This allele is of particular interest because it is very similar to HLADRBI*08 alleles (YSTGECY and lack of an associated HLA-DRB3 gene), but it lacks leucine at position 74, which is characteristic of all previously reported DRBI*08 alleles. The second HLA-DRBI*08 allele, DRBI*0804, differs from DRBI*0802 by a glycine to valine substitution at position 86. Human Immunology 34, 24-30 (1992)
HPMR
hyperpolymorphic region
INTRODUCTION H L A oligotyping, a method that utilizes sequence-specific oligonucleotide hybridization to detect polymorphic sequences [ 1 - 3 ] in amplified D N A , provides a practical means of routine H L A typing. Advantages of
* T h e names DRBI*0804 and DRBI*0805 were officially assigned to these new sequences by the W H O Nomenclature Committee in N o v e m b e r 1991. This follows the agreed policy that, subject to the condinons stated in the most recent Nomenclature Report [10], names will be assigned to new sequences as they are identified. Dsts of such new names will be published in the forthcoming W H O Nomenclature Report. The DRBI*0804 and DRBI*0805 sequences have been submitted to Gen Bank and assigned the accession numbers M844446 and M84357, respectively.
From the Blood Center of Southeastern Wisconsin and the Medical College of Wl~consin, Milu,aukee, Wisconsin. Address reprint requests to Dr. Lee Ann Baxter-Lou,e, The Blood Center of Southeastern Wisconsin, 1701 West Wisconszn Avenue, Milwaukee, WI 5.3233. Received August 15, 1991: aaeptedJanuary 2, 1992. 24 0198-8859/92/,$5.00
oligotyping include the ability to type a wide range of samples (e.g., nonviable cells and cells with low expression of H L A proteins) and the use of synthetic rather than biologic reagents. Another advantage of oligotyping is that specific amplification can reduce or eliminate ambiguity resulting from the sharing of a single polymorphic sequence by multiple alleles. This is accomplished by controlling the specificity of each amplification to generate amplified products of one locus, a particular subset of alleles, or an individual allele {4-7]. Analysis of a single allele or subset of alleles is essential for unambiguous assignment of oligotypes in certain heterozygous individuals. A method to oligotype all H L A - D R B 1 alleles that are associated with H L A - D R w 5 2 haplotypes has been reported [7]. H o w e v e r , due to the sharing of polymorphic sequences within this group, many heterozygous combinations cannot be resolved. This limitation was Human Immunology 34, 24-30 (1992) © American Society for Histocompatibility and lmmunogenetics, 1992
HLA-DRB l'D08/12 Alleles
25
TABLE 1 Primers utilized for HLA-DRB1*08/12 oligotyping Name
Sequence
Positive hybridization
PCR6 PCR5 PCR44 T7 M13forward
5'-ATTTCTTCAATGGGACGGAGC-3' 5'-CGCCGCTGCACTGTGAAGCTCTC-3' 5 '-GTACTCTACGGGTGAGTGTT-3' 5 '-AATACGACTCACTATAG-3 ' 5 '-GTTTTCCCAGTCACGACGT-3'
All DRB alleles All DRB alleles DRB 1*08, 12, 1404 pCR1000 vector pCR1000 vector
circumvented by developing an oligotyping method that utilizes a subset of HLA-DRw52 associated alleles containing YSTGECY (HLA-DRB 1*08/12/1404). Amplified DNA was hybridized to a panel of probes that differentiate all oligotypes associated with this subset of alleles. Using this method, two additional HLADRBI*08 alleles were detected and were confirmed by direct sequencing.
MATERIALS A N D M E T H O D S
Oligonucleotides.
Oligonucleotides were synthesized using either a Gene Assembler (Pharmacia Fine Chemicals, Piscataway, NJ) or a polymerase chain reaction (PCR) Mate (Applied Biosystems, Foster City, CA). The sequences of all primers and probes used in this study are provided in Tables 1 and 2. Probes were named as the polymorphic amino acid(s) encoded by the segment of the gene complementary to the oligonucleotide and a number corresponding to the position of the polymorphic amino acid(s) in the sequence. For example, L37L38 detects the nucleotides encoding two leucine residues found at positions 37 and 38 in DRBl*1201 and 1202.
TABLE 2
DRB codons 16-22 87-94 10-16
Oligotyping. Genomic DNA was isolated as described previously [8] from B-lymphoblastoid cell lines or white blood cells that were typed as HLA-DRw8 and -DRwI2 using serologic methods. Amplification was achieved using the PCR as described earlier [9] with the modifications described below. Intralocus-specific amplification utilized a 5' primer (PCR44) complementary to sequences encoding YSTGECY at positions 10-16 and a 3' primer (PCR5) complementary to codons 8794 of all HLA-DRB alleles (Table 1); MgCl2 was 3.5 mM; and thermal cycling was 20 seconds at 98°C, 30 seconds at 55°C, and 30 seconds at 72°C. Locus-specific amplification utilized a 5' primer (PCR6) that is complementary to most nucleotides at codons 16-22 of all HLA-DRB alleles and PCR5 (Table 1); MgC12 was 1.5 mM; and thermocycling was 15 seconds at 98°C, 20 seconds at 55°C, and 15 seconds at 72°C. Amplified DNA was hybridized with digoxigenin-labeled oligonucleotide probes as described earlier [9] using temperatures for probe hybridizations and washes that are listed in Table 2. The conditions necessary for obtaining the appropriate specificities of primers and probes were determined by oligotyping the B-lymphoblastoid cell lines that were characterized during the Tenth International Histocompatibility Workshop.
Oligonucleotide probes utilized for HLA-DRB1*08/12 oligotyping
Name
Codons
Sequence
L37L38 F37 $57 con6 la I67 F67 A74 L74 G86 V86
36-39 36-39 56-59 61-64 67-71 67-71 70-74 71-74 85-88 85-88
5'-CGCAGGAGCTCC-3' 5'-CGCACGAACTCC-3' 5'-CTCGGCGCTAGG-3' 5 '-TCTGGCTGTTCC-3' 5'-CCTGTCTTCCAGGATG-3' 5'-CTGTCTTCCAGGAAG-3' 5'-CGCGGCCCGCCTGT-3' 5'-CAGGGCCCGCCT-3' 5'-CTCTCACCAACC-3' 5'-CTCTCCACAACC-3'
con61 is the consensus probe.
Positive hybridization "1201, 1202 * 1404 "0801, 0803, All DRB 1 *0803, 1201 "0801, 0802, *0805 "0801, 0802, "0801, 0802, "1404, 0804
0805
0804, 0805, 1202 0803, 0804 0803, 0805
Hybridization temp (°C)
Wash temp
30 35 30 30 35 35 30 30 30 30
32 35 30 30 55 45 48 32 32 32
(°c)
26
M. Eberle and L. A. Baxter-Lowe
6
10
20
30
40
60
70
80
90
RFLE" - TSECHFFMP,TERVRFLDRYFYMQEEYVRFDSOVGEYRAVTELGRPOAEYt,AISGi(DLLEDRRAAVI)TYCRHIIYGWESFiVQRR ( .... vs-6--, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s . . . . . . . . . F . . . . . . . . . . . . . . . . . . s ........ ) c .... vs-~--v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s ......... F ...... , . . . . . . . . . . . ~. . . . . . . . . ) ( .... vs-~--Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F ...... L . . . . . . . . . . . ~ ........ ) ( .... ,S-6--, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *: . . . . . . , . . . . . . . . . . . . . . . . . . . . ) ( .... YS'G-'Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S ......... I ...... L ........... G ........ ) c .... v s - ~ - - v . . . . . . . . . . . . . . . . .... F . . . . . . . . . . . . . . . . . . . A--. ......... R---E . . . . . . . . . . . . . . . . . . . . ) ( .... YS-G- -Y ......... Y-E-H-H .... LL ........ F ......... V- -S ...... l ................. A ......... ) ( .... YS-G--Y ......... Y-E-H-H .... LL ........ F ......... V--S ...... F ................. A ......... )
colrisensus
DRBI*0805 DRBI*0801 DRBI*0802 DItBI*0804 DRBI*0803 DRB1*1404 DRB1*1201 DRB1*1202
F I G U R E 1 Alignmen~ of HLA-DRB1*08/12/1404 alleles. The amino acid sequences of DRB1 alleles detected in this assay (reviewed in Marsh and Bodmer [23]) are listed with the HLA-DRB consensus sequence at the top. Dashes indicate residues that are identical to the consensus sequence and the single-letter amino acid code shows deviation from the consensus sequence.
Products from alleles e n c o d i n g Y S T G E C Y (HLAD R B 1 * 0 8 / 1 2 ) were h y b r i d i z e d with a panel o f probes (Table 2) that detect p o l y m o r p h i c s e q u e n c e s that differentiate all r e p o r t e d alleles of this g r o u p (Fig. 1 and Table 3). I n t e r n a l controls, which are used to m o n i t o r the specificity o f each hybridization, were amplified using p r i m e r s P C R 6 and P C R 5 , which are specific for H L A D R B (Table 1). A c o n s e n s u s p r o b e (Table 2), which hybridizes to all H L A - D R B alleles, was i n c l u d e d to m o n i t o r the a m o u n t o f D N A b o u n d to the m e m b r a n e . N o m e n c l a t u r e for oligotypes is based u p o n the designations for H L A alleles that have b e e n r e p o r t e d by the W H O N o m e n c l a t u r e C o m m i t t e e for Factors of the H L A System [10]. O l i g o t y p e s are distinguished from alleles by s u b s t i t u t i n g a dash (-) for the asterisk (*).
Nucleotide sequencing of alleles. T h e D R B 1"08/12/1404specific 5' p r i m e r ( P C R 4 4 ) was utilized to amplify the
TABLE 3
50
second e x o n (codons 1 6 - 9 4 ) o f the H L A - D R alleles selectively. Amplified D N A was ligated into the p C R 1 0 0 0 vector ( I n v i t r o g e n C o r p o r a t i o n , San Diego, CA). Plasmid D N A was isolated from clear colonies and s e q u e n c e s of the inserts from m u l t i p l e clones were det e r m i n e d using vector p r i m e r s and standard dideoxy t e r m i n a t i o n m e t h o d s [11].
Serologic HLA typing. H L A serologic typing using microcytotoxicity assays [12] was g e n e r o u s l y p r o v i d e d by the H i s t o c o m p a t i b i l i t y and I m m u n o g e n e t i c s Laboratory o f the B l o o d C e n t e r of S o u t h e a s t e r n Wisconsin. T h e serologic specificities for samples c o n t a i n i n g the alleles designated D R B I * 0 8 0 5 and D R B I * 0 8 0 4 were A 2 , - ; B27,44(4); Cw 1 ,w5; D R 4 , w 8 , w 5 2 , w 5 3 ; D Q w 4 , w 7 , and A30,33; B35,w42(6); Cw4,-; DR4,w8,w52,w53; D Q w 7 , w 8 , respectively.
RESULTS AND DISCUSSION
HLA-DRBI*08/12/1404 oligotyping. A nonradioactive oligotyping system was d e v e l o p e d to p r o v i d e a practical, c o m p l e t e system o f typing H L A - D R B 1 * 0 8 / 1 2 / 1 4 0 4 alleles. This subset of H L A - D R B 1 alleles was specifically amplified to r e d u c e the complexity of data interpreta-
H y b r i d i z a t i o n p a t t e r n s o b t a i n e d for H L A - D R B 1"08/12 oligotyping Oligotype
Probe
0805
L37L38 F37 $57 con61 167 F67 A74 L74 G86 V86
. . ÷ + . + + + -
0801 . .
0802 .
.
.
+
+
+ .
+ + -
1201
1202
+ + -
÷ + + -
÷ + +
-
-
.
. .
1404
.
+ .
+ . + + -
0803
.
.
÷ + .
0804
. ÷ +
+ + + . + + -
.
. +
-
HLA-DRB 1"D08/12 Alleles
27
Probe: L74
Probe: con61 A 1
J
B
C
A
D
B
C
D
ifiiqii~
;i~ii~ii~ ....
iiiii
7
...... i;i~ii!
....
7 r;;iiiiii
9
~,,~®, i!iil; i!!!ii i,
9
lO
.............. iii!i~ !i!,;i!!;~:: ..... :ii)!ii
lO
11
i~
11
.... iiiii:i;/
FIGURE 2 Primary data from DRBl*08/12 oligotyping. A digoxigenin-labeled probe, con61, is a consensus probe that is utilized to monitor loading of DNA on membranes. Probe L74 detects the polymorphism (CTG) at codon 74. (A) Control DNA was amplified from the appropriate Tenth International Histocompatibility Workshop B-Lymphoblastoid Cell Lines and are included to monitor the specificity of hybridization. Primers (PCR6 and 5) were used to amplify all HLADRB alleles. (B-D) DNA from HLA-DRBl*08/12/1404 alleles was specifically amplified utilizing PCR44 and 5. Oligotypes were assigned as described in Materials and Methods. B7 was assigned an DRB1-0805 oligotype and thus lacks hybridization to L74. D6 is an ink dot used for orientation of membrane. Sample descriptions are provided in Table 5.
tion that results from sharing of certain polymorphic residues by multiple alleles. Selective amplification used a primer complementary to the nucleotides encoding Y S T G E C Y at positions 1 0 - 1 6 of this subset of alleles. Hybridization of the amplified D N A to a panel of ten nonradioactive probes permitted assignment of a unique oligotype to each reported allele in this group (Fig. 1): H L A - D R B I * 0 8 0 1 [13], 0802 [14, 15], 0803 [14], 0804 [7, 16], 1201 [17], and 1202 [18]. An additional allele, H L A - D R B l * 1 4 0 4 [19, 20] (assigned on "HLA-DRw8-iike" serologic specificity, classified as a member of the H L A - D R B I * 1 4 group) was also amplified using this primer and was detected in this assay. For the H L A - D R B l - 0 8 / 1 2 / 1 4 0 4 alleles, oligotypes are assigned based upon the specific pattern of hybridization (Table 3) with ten probes that detect the polymorphic sequences that distinguish these alleles from each other. For example, DRB1-0801 oligotype is as-
signed if the amplified D N A hybridizes with $57, F67; L74, and G86, and not to the remaining probes (i.e., L37L38, F37, I67, A74, and V86). Likewise, DRB11201 oligotype is assigned if hybridization was positive for L37L38 and I67 and negative for F37, $57, F67, A74, L74, G86, and V86. Figure 2 shows an example of two membranes (one with the consensus probe, con61, and one with L74) from a typical oligotyping assay. Assignment of precise oligotypes for many heterozygous individuals can be difficult due to the sharing of polymorphic residues among alleles. The probes used in oligotyping of the HLA-DRB1*08/12/1404 alleles can hybridize with many alleles, making specific amplification essential. When specific amplification of YSTGECY containing alleles is used, unequivocal assignment of oligotypes is possible for most combinations of alleles. The four combinations that cannot be differentiated are listed below: HLA-DRB 1"0801/0801 or - DRB 1"0801/0802 HLA-DRB 1"0805/0801 or - D R B 1"0805/0802 HLA-DRB 1"0801/0803 or - DRB 1"0802/0803 HLA-DRB1*0801/1201 or - D R B 1 * 0 8 0 3 / 1 2 0 2 This circumstance is uncommon because these alleles are present at a fairly low frequency [21]. A recent publication [7] describes oligotyping of alleles associated with the HLA-DRw52 supertypic group (DRB1*03/11/12/13/14/08) utilizing a primer that amplifies all alleles that encode EYST at positions 9-12. In this circumstance, at least 27 alleles are coamplified and the number of heterozygotes that cannot be resolved increases substantially. The oligotyping method described here provides a reliable, accurate method for oligotyping samples containing HLA-DRBl*08/12/ 1404 alleles and has been used to type about 100 samples successfully (Table 4).
TABLE 4
N u m b e r of H L A - D R B 1-08/12/1404 oligotypes
HLA-DRB1 oligotype
Number of oligotypes
0801 0802 0803 0804 0805 1201 1202 1404
32 14 14 1 2 35 2 0
28
TABLE 5
M. Eberle and L. A. Baxter-Lowe
Samples s h o w n in Fig. 2 Oligotype'
Position* A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 Bll C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Cll D 1 D2 D3 D4 D5
Sample nameb 9010 9087 9034 9037 9057 9097 9096 9069 9075 CS 9038 M7 M10 Mll M12 M13 M14 M16 M20 M21 M22 M23 M28 M29 M31 M32 M33 M34 M39 M44 M48 M52 M54 M56 M57 M58 M59 M60
AMAI STEINLIN SAVC SWEIG TEM EMJ LBF MADURa DKB BM16
DRB11501 0301 0401 1101 1401 1302 0702 0801 0901 0701, 1001 1202 07, 12 04, 12 04, 12 15/16, 08 07, 12 01, 12 04, 08 14, 08 07, 08 04, 08 08, 11 01, 08 07, 08 03, 12 03, 08 03, 08 07, 08 03, 08 03, 08 07, 08 08, 11 08, 10 15/16, 08 08, 11 15/16, 08 08, 12 03, 08
DRB3-
DRB4-
DRB50101
0101 0101 0202 0201 0301 0101 0101 0101 0202 X
X
X X
X X
X X
X
X
X X X X X X X X X X X X X X X X X X X
Position of samples shown in Fig. 2. bSamples with four digit number preceding name are from the Tenth International Histocompatibility Workshop. Those preceded with an M are samples of indicated primary oligotypes. cOligotypes were determined to identify groups of alleles using an HLA-DR oligotyping method that approximates serologic specificities [29]. An X represents the detection of a polymorphic sequence indicative of the locus.
Discovery of novel HLA-DRB1 alleles. H y b r i d i z a t i o n patterns p r e s e n t e d in T a b l e 3 d e m o n s t r a t e the d e t e c t i o n o f u n k n o w n H L A - D R alleles using oligotyping. T h e s e alleles are revealed by the p r e s e n c e o f c o m b i n a t i o n o f p o l y m o r p h i c s e q u e n c e s that are n o t a t t r i b u t a b l e to any p r e v i o u s l y r e p o r t e d alleles. S e q u e n c e analysis o f the exon e n c o d i n g the first d o m a i n c o n f i r m e d the seq u e n c e s p r e d i c t e d by o l i g o t y p i n g (Fig. 1). T h e first allele is identical to D R B I * 0 8 0 1 except that
c o d o n 74 (Fig. 1) is G C G (alanine) rather than C T G (leucine). This s u b s t i t u t i o n is particularly i n t e r e s t i n g because l e u c i n e at p o s i t i o n 74 is characteristic o f H L A D R w 8 m o l e c u l e s and could be i n v o l v e d in the formation o f an e p i t o p e r e c o g n i z e d by H L A - D R w 8 - specific alloantisera. T h e only r e p o r t e d e x c e p t i o n is H L A D R B 1 * 1 4 0 3 , which e n c o d e s l e u c i n e at 74, but was rep o r t e d as a b l a n k by serologic typing [22]. T h e new allele H L A - D R B I * 0 8 0 5 lacks l e u c i n e at position 74,
HLA-DRB 1"D08/12 Alleles
but was serologically typed as H L A - D R w 8 . It does share two nonexclusive characteristics of other D R B I * 0 8 alleles: the lack of an associated H L A - D R B 3 allele and the presence of first H P M R sequences, Y S T G E C Y . It is difficult to understand the relationship between these two characteristics and the structural basis for the H L A - D R w 8 serologic specificity. The second allele H L A - D R B I * 0 8 0 4 differs from H L A - D R B I * 0 8 0 2 by a single amino acid substitution at codon 86, a G G T (glycine) to ( G T G ) valine (Fig. 1). This allele was also independently observed by others [7, 16]. This dimorphism is c o m m o n and has already been reported for several other H L A - D R B alleles, such as H L A - D R B l * 1 5 0 1 and 1502; D R B I * 0 4 0 4 and 0408; and DRB3*0201 and 0202 (reviewed by Marsh and B o d m e r [23]). It has been suggested that this position is important for T-cell recognition. For example, Lang et al. [24] suggest that position 86 is involved in formation of the H L A - D w l 3 and -Dw14 determinant recognized by a T-cell clone (E38). Several investigators [ 2 5 - 2 8 ] have implicated positions 86 (along with position 85) in forming H L A - D R 1 associated allodeterminants detected in cellular assays. The physiologic significance of this dimorphism at position 86 can now be tested using oligotyping systems such as that reported here in which each allele is independently examined.
Origin of the HLA-DR alleles. Each of the two alleles reported here differ from reported alleles by two-basepair differences (CT and G C at nucleic acids 2 0 7 - 2 0 8 of H L A - D R B I * 0 8 0 1 and G T to T G at 2 4 4 - 2 4 5 of HLA-DRB1*0802). G e n e conversion events provide one explanation for the origin of the new H L A - D R variants. The lack of an H L A - D R B 3 product in all H L A - D R B I * 0 8 alleles is consistent with derivation of each of these alleles from a c o m m o n ancestor with gene conversion events introducing polymorphic sequences.
Future of oligotyping classification. As additional H L A alleles continue to be discovered, it is becoming apparent that the classification of H L A alleles according to serologic specificities will become increasingly difficult. For example, the first three HLA-DRw8-associated alleles that were discovered could be distinguished from all other H L A - D R alleles by the presence of leucine at position 74, the lack of a linked H L A - D R B 3 product, and the presence of the sequence, Y S T G E C Y , in the first H P M R ( Y S T G E C Y is also found in D R B I * 1 2 alleles). At that time, it appeared that leucine at position 74 could provide the basis for assignment of an HLAD R B 1-08 oligotype and could be implicated in the formation of an HLA-DRw8-specific epitope. However, the discovery of additional alleles raises questions regarding these possibilities. For example, the HLA-
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D R B I * 0 8 0 5 allele lacks leucine at position 74, but shares the two other distinguishing characteristics of HLA-DRw8-associated alleles. Another allele that was reported as "HLA-DRw8-1ike," D R B 1" 1404 [ 19], has alanine at position 74, but differs from H L A - D R B 1"08 alleles by linkage with an H L A - D R B 3 product. This suggests that leucine at position 74 is not essential for assignment of H L A - D R w 8 . Perhaps the D R B I * 0 8 classification should be reserved for alleles that are not linked to an H L A - D R B 3 locus and that encode Y S T G E C Y in the first H P M R or H L A - D R B 1. This classification is appealing from a teleologic standpoint, but sharing of polymorphic sequences and heterozygosity would make it difficult to detect an H L A - D R B 1-08 oligotype when products of a locus-specific amplification are examined. H L A oligotyping, a method that utilizes sequencespecific oligonucleotide hybridization to detect specific sequences in amplified D N A , is a practical method for routine H L A typing that provides a level of resolution that surpasses serologic typing. This level of resolution may be very important for selection of donors for bone marrow transplantation and studies involving the role of H L A in autoimmune disease. ACKNOWLEDGMENTS
We thank Dr. Jack Gorski for a critical review of the manuscript and insightful discussion. We are grateful to Ufuk Asu and the members of the Histocompatibility and Immunogenetics Laboratory and the DNA Diagnostics Laboratory of the Blood Center of Southeastern Wiscdnsin for providing assistance in this investigation. This study was supported by the National Institutes of Health (R01 AI28034) and the Blood Center of Southeastern Wisconsin.
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