How Many Probes Are Needed for HLA-DPB1 Typing with Sequence-Specific Oligonudeodde Probes? A Theoretical Approach Using Computer Simulation Carlheinz R. Miiller and Thomas H. Eiermann
ABSTRACT: HLA-DP genotyping with sequencespecific oligonucleotides is used to detect known sequence variations in the polymorphic segments of the DPB1 second exon. This approach is a valuable method replacing the mdious cellular definition of DP polymorphism. We have addressed, by computer simulation, the question: what is the minimum number of probes needed to provide an unambiguous assignment of HLA-DP alleles by genotyping of heterozygous individuals? We were able to reduce the number of probes in a set defining the presently known 22 different alleles and most of
ABBREVIATIONS A$O allele-specific oligonucleotides HTC homozygous typing cells HLA human leukocyte antigens PCR polymerase chain reaction
the hererozygous combinations to 18 probes, Only two pairs of allelic combinations cannot be distinguished by this method, neither with our optimized set of probes nor with any larger set comprising probes of reasonable length. This is because two pairs of alleles may be the result of a reciprocal genetic exchange. These two pairs, however, could be distinguished by family analysis, direct sequencing, or DNA amplification using specific primers chosen from the polymorphic ends of the DPB1 second exon. Human Immunology 30, 2 2 - 6 (199;)
PLT RFLP SSO
primed lymphocyte typing restriction fragment length polymorphism sequence-specific oligonucleotides
INTRODUCTION Class l i human leukocyte antigens (HLA) are heterodimerit cell surface glycoproteins controlling the immune response by mediating the presentation of antigen to the T-cell receptor leading to specific activation of T lymphocyres [1, 2]. The study of their extensive allelic polymorphism plays an important role in tissue matching for organ transplantation [3, 4]. The DP antigens are part of the class II series of H L A antigens and were
IZmmthe Department uf TransJuffon Medicine, Uuil ersity of ulm attd the Department of Tramplantatlon Immunology. Red Cross Blood Bank. Ulm, F.R.G. Address reprint requests ta Dr. Thomas H. Eiemann. Red Cross Blond Bank. Helmholtzs~r. 10, 19-7900 Ulna. ER.G. Received ~ebruary 5, 1990; acceptedJuly 17, I990. 22
0198-885919tt$3.50
independently discovered by Shaw and Termijtelen by means of the primed lymphocyte typing (PLT) [5, 6]. Serologic analysis of DP antigens has been reported [7, 8], but it is hampered by the lack of suitable reagents. Cloning o f the DPA 1 and DPB 1 genes has allowed the use of restriction fragment length polymorplfism (RFLP) analysis as an alternative method [9-11]. Most recently, cloning and sequencing of the polymerase chain reaction (PCR)-amplified polymorphic second exon of D P B I locus has made it possible to use sequence-specific oligonudeotides (SSO) for the genotyping of D P molecules [ 12, 13]. This seems currently the most simple and promising approach for DP typing on a routine basis. Until now this method has been used mainly m type Human Immunology 30, 22-6 (1991) © Amedcan So¢ieW for Histo¢orat~tibility and Immunogenetics, 1991
HLA-DPBI Typing with SSO
a panel o f h o m o z y g o u s , in part, c o n s a n g u i n o u s typing cells ( H T C ) . U s i n g a set o f 14 d i f f e r e n t p r o b e s Angelini e t al. [ I 4 ] were able to define 13 different alleles in a p a n e l o f 45 H T C . H o w e v e r the p r o b l e m o f w h e t h e r an u n a m b i g u o u s typing o f D P - h e t e r o z y g o u s individuals is feasible and h o w m a n y p r o b e s are necessary to achieve this goal r e m a i n s u n r e s o l v e d . W e have a d d r e s s e d this p r o b l e m using a c o m p u t e r s i m u l a t i o n to analyze t h e i n f o r m a t i o n c o n t a i n e d in a set o f p r o b e s with r e s p e c t to a c o m p l e m e n t a r y set o f alleles and s u b s e q u e n t l y tried to r e d u c e this set. In this p a p e r w e will s h o w that a set o f t 8 S S O is sufficient to define t h e p r e s e n t l y k n o w n 22 different alleles and a m a x i m u m o f their possible h e t e r o z y g o u s c o m b i n a t i o n s .
MATERIAL
AND METHODS
The program minprobe. T h e p r o g r a m minprobe d e s c r i b e s t h e a m o u n t o f i n f o r m a t i o n that w e can obtain o n t h e p h e n o t y p e s o f a p o l y m o r p h i c g e n e by hybridization o f S S O to D N A amplified with p r i m e r s specific for that g e n e . F u r t h e r , it analyzes t h e r e d u n d a n c y c o n t a i n e d in a set o f S S O and s u g g e s t s p r o b e s that can be o m i t t e d w i t h o u t loss o f i n f o r m a t i o n . T h e i n p u t file d e s c r i b e s t h e h y b r i d i z a t i o n p a t t e r n s o f a s e t o f S S O with t h e c o r r e s p o n d i n g set o f alleles. T h e s e data are p r e s e n t e d in a plain A S C I I file c o n t a i n i n g essentially a r e c t a n g u l a r m a trix o f " + " and " - " c h a r a c t e r s with h e a d e r s for all rows a n d c o l u m n s . T h e reaction p a t t e r n o f a single p r o b e can b e c o n t a i n e d e i t h e r in a r o w o r in a c o l u m n , w h a t e v e r is m o r e c o n v e n i e n t with regard to t h e size o f b o t h sets. W e usually g e n e r a t e t h e s e files by m e a n s o f o t h e r prog r a m s (e.g., accessing o u r DNA database) b u t they can also be c r e a t e d by any text editor. Minprobe w o r k s in several phases: 1) T h e w h o l e i n p u t file is read. 2) T h e reaction patterns o f all the alleles are s e a r c h e d for subs e t s with identical patterns. T h e s e s u b s e t s are printed o u t a n d only o n e allele f r o m e a c h s u b s e t is r e t a i n e d for f u r t h e r analysis. 3) T h e reaction p a t t e r n s o f all h o m o z y g o u s and h e t e r o z y g o u s c o m b i n a t i o n s o f t h e r e m a i n i n g alleles are calculated and analyzed by a similar proced u r e as above. S u b s e t s o f t h e set o f all g e n o t y p e s s h o w ing t h e s a m e reaction p a t t e r n are t h e n p r i n t e d o u t and, again only o n e copy o f e a c h s e t is retained. 4) A recursive a l g o r i t h m is applied to e l i m i n a t e u n n e c e s s a r y (i.e., r e d u n d a n t ) p r o b e s . T h e p r o g r a m is written in C a n d runs on a SIEMENS PCD-3T (386/16MHz/5MBt und e r S C O - X E N I X - 3 8 6 . T h e binary is available o n 1 . 2 M B floppies in tar f o r m a t at a n o m i n a l charge. ( D u e to limitations o f t h e linear a d d r e s s space, the M S - D O S v e r s i o n o f t h e p r o g r a m m was u n a b l e to cope with the p r o b l e m d i s c u s s e d here.)
DPBI sequences. T h e 22 d i f f e r e n t D P B I s e q u e n c e s w h i c h w e r e u s e d to g e n e r a t e t h e hybridization matrix to
23
calculate the references D P B 1"0202, DPBI~0501, DPBI*090I, (DPwlb); DPBI~I901); DPBI'IS(H, (DPB2).
o p t i m i z e d set o f S S O were taken f r o m [12} ( D P B 1 ~0101, DPBI~020I, D F B l ~0301., D P B 1 ~0-~0 I, D P B I "0402, DPBI~0601, DPBt~0701, DPBI*0ggL DPBI~t001, DPBl~llOIk [15.3 [13~ CDPB]~ 1 3 0 l , DPBI~IS{H, [16] f D P B t ~ 1201, DPBI~1401, D P B I * 1 6 0 1 , D P B I ~ I T 0 1 } ; and [17]
RESULTS AND DISCUSSION T h e se~ o f 14 D P B I second-exoa-specific oligonucleotides used by Angelini ct al. [ t 4] to define l 5 differe n t D P B 1 alleles was tested for its ability to resolve all possible h e t e r o z y g o u s c o m b i n a t i o n s (Table 1). C o m p u t e r s i m u l a t i o n using t h e p r o g r a m mi~prnhe sb.owed that this set o f SSO could be able to discriminate bet w e e n all h o m o z y g o u s a n d m o s t h e t e r o z y g o u s alle~ic c o m b i n a t i o n s , b u t ir c a n n o t differentiate a panel o f eight h e t e r o z y g o u s g e n o t y p e s . W e f o u n d f o u r pairs o f h e t e r o z y g o u s variants s h o w i n g identical hybridization p a t t e r n s with t h e 14 different S S O ~Table t). [n addition, w e o b s e r v e d that t h e s e c o n d S S O p r o b e in this set ( D B l I) was r e d u n d a n t and could be omitted. N e x t , we tried to find h o w m a n y S S O p r o b e s have to be p r e p a r e d to resolve all possible a|lelic c o m b i n a t i o n s g i v e n hy t h e p r e s e n t l y k n o w n 22 different D P B t alleles. First, f r o m all t h e c u r r e n t l y available s e q u e n c e s o f t h e D P B 1 s e c o n d e x o n , we g e n e r a t e d 101 S S O to g e t h e r with t h e i r reaction p a t t e r n s with t h e s e alieles u s i n g o u r p r o g r a m genprobe (not to be described h e r e in detail). T h e s e are all possible i n f o r m a t i v e SSO with l e n g t h s b e t w e e n 10 a n d 23 bases and n o p o l y m o r p h i s m s at t e r m i n a t i n g positions. W e t h e n eliminated p r o b e s hybridizing also with D P B 2 a n d duplicate copies o f p r o b e s s h o w i n g identical reaction patterns. T h e r e s u l t i n g reaction matrix with 4 6 p r o b e s left was t h e n p r e s e n t e d to the p r o g r a m minpro&.
TABLE
O u t p u t o f t h e p r o g r a m minpmbe u s i n g t h e D P B 1-specific oligonucleorides f r o m r e f e r e n c e 14
1
The folhnwing hererozygtms combinarmns cannot he distinguished: 8 genoD'pes in .i groups with up to 2 identical combinations + -
--',-
+
-
-
-
+ -
-
+ . . . . "~~ ++ +- - - + - ÷ - + +- + - - - -~- + - + + - + .... + + - + + .... + .... + +- + + +--++ ~ + .... + F- - + - -
+ +
DPBl'04fll
++ ++ ++ ++ F ~+4 ++
DPBI'02111 DPB i *0202 DPBi "{bif I DPP~| "0402 DPBI'O21II DPBI'02UI DPBI "021)2
DPBI'0801 DPIBl~070! DPBP070! DPBI" 1901 DPB 1" 1901 DPBI'050t DPBI~IgOt DPBI~0801
24
C.R. Miiller and T. H. Eiermann
TABLE 2
Reaction patterns of an optimized set of SSO defining D P B I genotypes $SO
Allele
O1
DPBI'OIOI
+
DPBI'0201
-
DPB
i'0202
.
DPBIb
-
DPB
1 * 0401
.
DPB
1 *0402
-
DPB
I "0501
DPB
1 ~0701
DPB
.
.
.
.
.
.
-
DPBI*1101
.
D P B I * I 4 0 1
.
DPBI'ISOI
+
D P B [ ' 1 6 0 1
-
DPB['I70[
.
DPBI'Ig0i
180l
.
. .
.
-
+
+
.
+
+
.
+
+
+
-
+
+
+
+
.
+
+
+
+
.
-
-
-
+
-
-
+
-
+
.
+
+
-
_
.
. + .
. +
. -
.
.
.
•
-
. +
We found that all informatiun accessible by this method could already be gained using a subset of only 18 SSO. The reaction patterns o f these probes are shown in Table 2. Since many o f the reaction patterns can be obtained by more than one probe, we are curremly investigating which o f these probes shows the best handling under laboratory conditions. For example, it is possible to choose a probe of length 18 for each of the reaction patterns listed in Table 2. At first glance, this result might seem surprising since it shows that the number of SSO probes can be fewer than the number of alleles to be defined. It also means that selecting allele-specific oligonucleotide (ASO) probes, as originally suggested [18], is not the optimal strategy even when possible. Moreover, it is not always possible to find probes specific for each allele, since it is known that the polymorphism of class II genes is in part a patchwork structure of sequence differences generated by gene conversion [19-21]. Reducing the number of probes will be essential for effective work in tissue typing laboratories. For that purpose selecting SSO reacting with a number of alleles is preferable, since the maximum information for typing (usually heterozygous) cells is contained in a probe reacting with about (a + I/2) • (1 - 1/V'2) alleles out of a set of a alleles. Of course, in most cases the smallest defining subset of probes is far from being unique. An exhaustive
.
+
.
+
-
.
.
.
.
.
.
.
. -
+
.
-
-
-
. -
+
-
-
+
-
-
-
+
+
+
-
+
+
+
-
+
+
+
+
+
+
+
+
-
-
q-
-
+
-
+
+
+
+
-
+
+
+
-
-
-
.
+
-
-
÷
. -
+
+
-
-
-
+
-
+
-
-
-
+
+
+
+
.
-
-
. +
.
+
-
.
.
.
-
.
.
.
-
+
.
.
-
.
-
. +
+
.
.
.
+
.
.
.
.
.
+
.
+ .
.
-
+
.
+ .
.
.
.
.
+
. -
.
.
.
.
.
-
+
+ .
.
.
+
18
+
. .
-
-
÷
.
+
-
17
+
-v
-
.
16
.
+
.
+
+
+
+
.
+
+
-
.
.
-
+
.
+
+
+
-
.
-
+ -
-
.
-
-
+
.
+
+
.
.
÷
-
+
.
15
+
.
.
14
+
-r
.
13
-
+
+
.
-
12
.
.
.
.
.
.
.
11
-
-
.
+
-
-
+
+
"~
.
.
+
+ .
+
.
.
.
l0
.
-
+
DPBI°I30I
+
.
.
.
.
-
.
.
. -
1 "0901
DPBI"
.
07
-
.
+
.
09
. .
-
.
DPBI'I201
+
. -
08
. +
.
+
.
06
+
. -
-
. +
.
.
.
05
.
-
.
-
DPBIOl0Ol
04
.
.
DPB|'0801
03
-
.
DPBI'0301
DPBI*0601
G2
.
. -
.
.
+
search for the minimum means traversing the "tree of subsets" o f probes down to each point where a loss of information occurs. Let a denote the number of alleles, p the number of probes, and f ( 0 < f < 1) the fraction of probes that can be omitted, then the computing time cost for a procedure like that can be estimated by the expression l f~2I- I,-v; P(p, a ) 2 P ~ J-v; e-I~-'I~dx where P(p, ~) is some function dominated by a polynomial of order 4 in a and I in p and therefn re nor contributing significantly to the tremendous growth of this expression. However, assuming the other variables constant, the rest o f the expression shows an exponential behaviour with respect to p. For small values o f f , an even more dramatic growth with respect to the number of redundant probes is found. Therefore, with increasing size and redundancy of the set of probes the search can become extremely time consuming. In sets of probes, such as the one examined here, we cannot be sure that the number of probes at the time we stopped our search is definitely the minimum. Our computer simulation revealed another interesting fact. There are two pairs of hererozygous genotypes which are not separable even using all possible SSO (Table 3). Again, as observed before (Table 1), ceils possessing the DPBI~0401, D P B I ' 0 8 1 0 phenotype
HLA-DPB1
TABLE 3
Typing
with SSO
25
DPB[ second exons of hererozygous alielic combinations showing identical hybridization pa~terns with the set of probes from Table 2
DBPI*080t DPB 1*0401
............................
DPB i *0201
..................................................................
DPI3 ! "0701
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A .................
.
.
_
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
AA . . . . . . . . . .
K ......
M.......
GGP~
M ....... .
.
.
.
.
.
.
.
.
.
K
.AA . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
GGP~ .
.
.
.
.
DBPI'0801 DPBI'I701
VH.L ........................................
D ................
VFI,L ...........................................
D .............................
~ ...........
DPB1"1601 D P B 1" 0 9 0 [
OniF pol~'mot~hie nesidues (amino ~cids no. 8 - 8 7 ) different from D P B I ' 0 8 0 1 ar~ ~hnwn T h e braces marked with ° denote regions where crosslng-o,~er could here r=ken place. braces marked with T denote regions where specific primers ¢an be chosen m distinguish these ge~ozy~es.
showed an identical hybridization pattern to DPBI*020 I, DPB l*0701-posifive cells. In contrast, by additional SSO, the other three pairs of nonseparable variants observed previously (Table 1) could be differentiated. Inclusion of recently described alleles (DPvclb, DPBI*t20[, -DPBI*[801) reveated an additional pair of phenotypes (DPB 1"090 l, DPB 1=1601 and DPB ! "0801, DPB 1~t 701; Table 3) with identical hybridization patterns even with the optimized set of SSO. Table 3 illustrates the reason why these situations occur, In both cases one pair of alleles (e.g., DPBI~0701 and DPBI*020I) can be generated by reciprocal crossing-over between the other pair of alleles (e.g., DPBI~0401 and DPBI~0801). Only SSO probes taken from the crossing-over area would solve the problem. But since the crossing-over area spans 24 bp and 60 bp of DNA with two mismatches at the end of the probes, suitable hybridization conditions are unlikely to be attained [22]. Therefore, some individuals cannot be definitely typed by simple genotyping. This may be relevant for typing of donor pools for unrelated bone marrow transplantation, where family typing seems rather impractical to resolve ambiguous phenotypes. Alternative methods to resolve such ambiguous phenorypes ~tould be to perform a DNA amplification using primers chosen from the polymorphic ends of the DPB[ second exon (Table 3) [23]. This would allow amplification of only one of the two alleles involved in each phenotype. Then these alleles can be distinguished by hybridization with a single probe. Extending this method could even resolve double crossing-over situations involving three genes with large homologous parts between the polymorphisms enclosing the crossing-
over sites. Currently this still appears to be less work than sequencing the amplified DNA of the cell. CONCLUSIONS In conclusion we have shown that computer simulation is a valuable tool to optimize sets of 550 for HLA-DP typing, it has been ~ble to detect previously unknown equivocal patterns of hybridization by variant heterozygous phenotypes in a recendy published series of oiigonucleotides. We were ~bie to define an optimized s e t of oligonucleotides which allows the definition of 72 alleles using only 18 different SSO. Testing the vM~e of these probes in practical use is unde¢ way ~nd hybridization and washing conditions of these probes w i l l be detailed elsewhere~ Moreover minprobeshowed the limkalion of this new genoryping method given by the f~ct that some alleles may be the result of reciprocal crossing-over events generating phenotypes not separable by simple genotyping of DPB1 genes.
ACKNOWLE'DGM ~NT$
We thank M. Schimmer for writing our DNA editor and m a i n t a i n i n g o u r DNA database and !. Rich for critk~ reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschaft SFB 322 C5. REFERENCES 1. D a v i s M M ,
Btorkman
PJ: T-ceil dntigen
receptor
genes
and T-ceU recognition. Nature 334:395, 1988. 2. Brown JH, J~de~zky T, Saper MA, Samr=oul 8, Bjorkman PJ, Wiles CD: A hypothetical mode[ of the
26
foreign antigen binding site of class 11 histocompatibility molecules. Nature 322:845, 1988. 3. Odum N, Pla:z P, Jakobsen BK, Petersen CM, Jacobsen N, Moiler J. Ryder LP, Lamm L, Sveigaard A: HLA-DP and bone marrow transplantation: DP-incompatibility and severe acute graft versus host disease. Tissue Antigens 30:213, 1987. 4. Amar A, Nepom GT, Mickelson EM, Erlich HA, I lansen JA: HLA-DP and HLA-DO genes in presumptive HLAidentical siblings: Structural and functional identification of altelic variation. J Immunol 138:1947, 1987. 5. Shaw S, Johnson AH, Shearer GM: Evidence for a new segregant series of B-cell antigens that are encodcM in the HLA-D region and that stimulate secondary allogeneic proliferative and cytotoxic responses. J Exp Med 152:565, 1980. 6. Termiitelen A, Bradley BA, van Rood Jj: A new determinant defined by PLT, coded for in the HLA region and apparently independent of the HLA-D and DR loci. Tissue Antigens 15:267, 1980. 7_ Tanigaki N, Tosi RM, Parodi B, Snrrentino R, Ferrara GB, Strominger JL: Detection of HLA-DP serological allodeterminants by the use of radio-iodinated DP molecules. Eur J lmmunol 17:743, 1987. 8. Mueller-Eckhardt G, Kiefel V, Schmidt A, Tlusty A, Santoso S, MueUer~Eckhardt C: Discrimination of antibodies against angitens of different MHC loci in human sera by monodonal antibody-specific immobilization of leukocyte antigens. Hum Immunol 25:125, 1989. 9. Bodmer JG, Bodmer WF, Heyes J, So AK, Tonks S, Trowsdale J. JongJ: Identification of HLA-DP polymorphism with DP a and DP # probes and monoclonal antibodies: Correlation with primed lymphocyte typing, Proc Nad Acad Sci USA 84:45r'6, 1987. 10. Hyldig-Nielson JJ, Morling N, Odum N, Ryder LP, Platz P, Jakobsen B, Svejgaard A: Restriction fragment length polymorphism of rhe HLA-DP subregion and correlation of HLA-DP phenotypes. Proc Natl Acad Sci USA 84:1644, 1987. I 1. Maeda M, Inoko H, Andn A, Uryu N, Nagata Y, Tsuji K: HLA-DP typing by analysis of DNA restriction fragment length polymorphisms in the HLA-DP/3 subregion, Hum lmmunol 21:239, 1988. 12. Bugawan TL, Horn GT, Long CM, Mickelson EM, Hunsen JA, Ferrara GB, Angelini G, Erlich HA: Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP-a and DP-fl loci. J lmmunol 1,~h4024, 1988.
C.R. Miilfer and T. 1-t. Eiermann
13. Bugawan TL, Angelini G, Larrick J, Auricchio S, Ferrara GB, Erlich HA: A combination of a particular HLA-DP's allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease. Nature 339:470, 1989. 14. Angelini G, Bugawan TL, Delfino L, Erlich HA, Ferrara GB: HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides. Hu.~ lmmunol 26:169, 1989. 15. Lee JS, Sarmrius S, Briat.~ P, Choi E, Cullen C, Lepaslier D, Yunis EJ: Sequence polymorphism of HLA-DP fl chains. Immunogenetics 29:346, 1989. 16. 8egnvich AB, BugawauTL, Nepom BS, Klitz W, Nepom GT, Erlich HA" A specific HLA-DP fl allele is associated with pauciarticular rheumarhoJd arthritis but not adult rheumathoid arthritis. Proc Natl Acad Sci USA 86:9489, 1989. 17. Gustafsson K, Widmark E, Jonsson A-K, Servenius B, Sachs DH, Larhamm~r D, Rask L, Peterson PA: Class 11 genes of the human maior histocompatibilky complex-evolution of the DP region as deduced from nucleotide sequences of the four genes. J Biol Chem 262:8778, 1987. 18. Salki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA: Analysis of enzymatically amplified fl-globin and HLA DQ a DNA with allele specific probes. Nature 32:163, 1986. 19. Angelini G, De Preval C, GorskiJ, Math B: High resolution analysis of the human HLA-DR potymorphism by hybridization with sequence-specific oligonucleotide probes. Proc Narl Acad Sci USA 83:4489, 1986. 20. Gorski J, Math B: Polymorphism of human ia antigens: Gene conversion between two DR's loci results in a new HLA-D/DR specificity. Nature 322:67, 1986. 21. Tiercy J-M, Gorski J, Betuel H, Freidel AC, Gebuhrer L, jeannet M, Math B: DNA typing of DRw6 subtypes: Correlation with DRB1 and DRB3 allelic sequences by hybridization with oligonucleotide probes. Hum Immunol 24:l, 1989. 22. lkuta S, Tagaki K, Wallace RB, hakura K: Dissociation kinetics of 19 base paired olignnucleotide-DNA duplexes containing different single mismatched base pairs, Nucleic Acids Res 15:797, 1987. 23. Shaft S, Saiki R, Erlich H: New Methodology fo~ HLA class I1 oligoaucleotide typing using polymerase chain reaction (PCR) amplification. Hum lmmunol 23:143, 1988.
ABSTRACT: HLA-DP genotyping with sequencespecific oligonucleotides is used to detect known sequence variations in the polymorphic segments of the DPB1 second exon. This approach is a valuable method replacing the mdious cellular definition of DP polymorphism. We have addressed, by computer simulation, the question: what is the minimum number of probes needed to provide an unambiguous assignment of HLA-DP alleles by genotyping of heterozygous individuals? We were able to reduce the number of probes in a set defining the presently known 22 different alleles and most of
ABBREVIATIONS A$O allele-specific oligonucleotides HTC homozygous typing cells HLA human leukocyte antigens PCR polymerase chain reaction
the hererozygous combinations to 18 probes, Only two pairs of allelic combinations cannot be distinguished by this method, neither with our optimized set of probes nor with any larger set comprising probes of reasonable length. This is because two pairs of alleles may be the result of a reciprocal genetic exchange. These two pairs, however, could be distinguished by family analysis, direct sequencing, or DNA amplification using specific primers chosen from the polymorphic ends of the DPB1 second exon. Human Immunology 30, 2 2 - 6 (199;)
PLT RFLP SSO
primed lymphocyte typing restriction fragment length polymorphism sequence-specific oligonucleotides
INTRODUCTION Class l i human leukocyte antigens (HLA) are heterodimerit cell surface glycoproteins controlling the immune response by mediating the presentation of antigen to the T-cell receptor leading to specific activation of T lymphocyres [1, 2]. The study of their extensive allelic polymorphism plays an important role in tissue matching for organ transplantation [3, 4]. The DP antigens are part of the class II series of H L A antigens and were
IZmmthe Department uf TransJuffon Medicine, Uuil ersity of ulm attd the Department of Tramplantatlon Immunology. Red Cross Blood Bank. Ulm, F.R.G. Address reprint requests ta Dr. Thomas H. Eiemann. Red Cross Blond Bank. Helmholtzs~r. 10, 19-7900 Ulna. ER.G. Received ~ebruary 5, 1990; acceptedJuly 17, I990. 22
0198-885919tt$3.50
independently discovered by Shaw and Termijtelen by means of the primed lymphocyte typing (PLT) [5, 6]. Serologic analysis of DP antigens has been reported [7, 8], but it is hampered by the lack of suitable reagents. Cloning o f the DPA 1 and DPB 1 genes has allowed the use of restriction fragment length polymorplfism (RFLP) analysis as an alternative method [9-11]. Most recently, cloning and sequencing of the polymerase chain reaction (PCR)-amplified polymorphic second exon of D P B I locus has made it possible to use sequence-specific oligonudeotides (SSO) for the genotyping of D P molecules [ 12, 13]. This seems currently the most simple and promising approach for DP typing on a routine basis. Until now this method has been used mainly m type Human Immunology 30, 22-6 (1991) © Amedcan So¢ieW for Histo¢orat~tibility and Immunogenetics, 1991
HLA-DPBI Typing with SSO
a panel o f h o m o z y g o u s , in part, c o n s a n g u i n o u s typing cells ( H T C ) . U s i n g a set o f 14 d i f f e r e n t p r o b e s Angelini e t al. [ I 4 ] were able to define 13 different alleles in a p a n e l o f 45 H T C . H o w e v e r the p r o b l e m o f w h e t h e r an u n a m b i g u o u s typing o f D P - h e t e r o z y g o u s individuals is feasible and h o w m a n y p r o b e s are necessary to achieve this goal r e m a i n s u n r e s o l v e d . W e have a d d r e s s e d this p r o b l e m using a c o m p u t e r s i m u l a t i o n to analyze t h e i n f o r m a t i o n c o n t a i n e d in a set o f p r o b e s with r e s p e c t to a c o m p l e m e n t a r y set o f alleles and s u b s e q u e n t l y tried to r e d u c e this set. In this p a p e r w e will s h o w that a set o f t 8 S S O is sufficient to define t h e p r e s e n t l y k n o w n 22 different alleles and a m a x i m u m o f their possible h e t e r o z y g o u s c o m b i n a t i o n s .
MATERIAL
AND METHODS
The program minprobe. T h e p r o g r a m minprobe d e s c r i b e s t h e a m o u n t o f i n f o r m a t i o n that w e can obtain o n t h e p h e n o t y p e s o f a p o l y m o r p h i c g e n e by hybridization o f S S O to D N A amplified with p r i m e r s specific for that g e n e . F u r t h e r , it analyzes t h e r e d u n d a n c y c o n t a i n e d in a set o f S S O and s u g g e s t s p r o b e s that can be o m i t t e d w i t h o u t loss o f i n f o r m a t i o n . T h e i n p u t file d e s c r i b e s t h e h y b r i d i z a t i o n p a t t e r n s o f a s e t o f S S O with t h e c o r r e s p o n d i n g set o f alleles. T h e s e data are p r e s e n t e d in a plain A S C I I file c o n t a i n i n g essentially a r e c t a n g u l a r m a trix o f " + " and " - " c h a r a c t e r s with h e a d e r s for all rows a n d c o l u m n s . T h e reaction p a t t e r n o f a single p r o b e can b e c o n t a i n e d e i t h e r in a r o w o r in a c o l u m n , w h a t e v e r is m o r e c o n v e n i e n t with regard to t h e size o f b o t h sets. W e usually g e n e r a t e t h e s e files by m e a n s o f o t h e r prog r a m s (e.g., accessing o u r DNA database) b u t they can also be c r e a t e d by any text editor. Minprobe w o r k s in several phases: 1) T h e w h o l e i n p u t file is read. 2) T h e reaction patterns o f all the alleles are s e a r c h e d for subs e t s with identical patterns. T h e s e s u b s e t s are printed o u t a n d only o n e allele f r o m e a c h s u b s e t is r e t a i n e d for f u r t h e r analysis. 3) T h e reaction p a t t e r n s o f all h o m o z y g o u s and h e t e r o z y g o u s c o m b i n a t i o n s o f t h e r e m a i n i n g alleles are calculated and analyzed by a similar proced u r e as above. S u b s e t s o f t h e set o f all g e n o t y p e s s h o w ing t h e s a m e reaction p a t t e r n are t h e n p r i n t e d o u t and, again only o n e copy o f e a c h s e t is retained. 4) A recursive a l g o r i t h m is applied to e l i m i n a t e u n n e c e s s a r y (i.e., r e d u n d a n t ) p r o b e s . T h e p r o g r a m is written in C a n d runs on a SIEMENS PCD-3T (386/16MHz/5MBt und e r S C O - X E N I X - 3 8 6 . T h e binary is available o n 1 . 2 M B floppies in tar f o r m a t at a n o m i n a l charge. ( D u e to limitations o f t h e linear a d d r e s s space, the M S - D O S v e r s i o n o f t h e p r o g r a m m was u n a b l e to cope with the p r o b l e m d i s c u s s e d here.)
DPBI sequences. T h e 22 d i f f e r e n t D P B I s e q u e n c e s w h i c h w e r e u s e d to g e n e r a t e t h e hybridization matrix to
23
calculate the references D P B 1"0202, DPBI~0501, DPBI*090I, (DPwlb); DPBI~I901); DPBI'IS(H, (DPB2).
o p t i m i z e d set o f S S O were taken f r o m [12} ( D P B 1 ~0101, DPBI~020I, D F B l ~0301., D P B 1 ~0-~0 I, D P B I "0402, DPBI~0601, DPBt~0701, DPBI*0ggL DPBI~t001, DPBl~llOIk [15.3 [13~ CDPB]~ 1 3 0 l , DPBI~IS{H, [16] f D P B t ~ 1201, DPBI~1401, D P B I * 1 6 0 1 , D P B I ~ I T 0 1 } ; and [17]
RESULTS AND DISCUSSION T h e se~ o f 14 D P B I second-exoa-specific oligonucleotides used by Angelini ct al. [ t 4] to define l 5 differe n t D P B 1 alleles was tested for its ability to resolve all possible h e t e r o z y g o u s c o m b i n a t i o n s (Table 1). C o m p u t e r s i m u l a t i o n using t h e p r o g r a m mi~prnhe sb.owed that this set o f SSO could be able to discriminate bet w e e n all h o m o z y g o u s a n d m o s t h e t e r o z y g o u s alle~ic c o m b i n a t i o n s , b u t ir c a n n o t differentiate a panel o f eight h e t e r o z y g o u s g e n o t y p e s . W e f o u n d f o u r pairs o f h e t e r o z y g o u s variants s h o w i n g identical hybridization p a t t e r n s with t h e 14 different S S O ~Table t). [n addition, w e o b s e r v e d that t h e s e c o n d S S O p r o b e in this set ( D B l I) was r e d u n d a n t and could be omitted. N e x t , we tried to find h o w m a n y S S O p r o b e s have to be p r e p a r e d to resolve all possible a|lelic c o m b i n a t i o n s g i v e n hy t h e p r e s e n t l y k n o w n 22 different D P B t alleles. First, f r o m all t h e c u r r e n t l y available s e q u e n c e s o f t h e D P B 1 s e c o n d e x o n , we g e n e r a t e d 101 S S O to g e t h e r with t h e i r reaction p a t t e r n s with t h e s e alieles u s i n g o u r p r o g r a m genprobe (not to be described h e r e in detail). T h e s e are all possible i n f o r m a t i v e SSO with l e n g t h s b e t w e e n 10 a n d 23 bases and n o p o l y m o r p h i s m s at t e r m i n a t i n g positions. W e t h e n eliminated p r o b e s hybridizing also with D P B 2 a n d duplicate copies o f p r o b e s s h o w i n g identical reaction patterns. T h e r e s u l t i n g reaction matrix with 4 6 p r o b e s left was t h e n p r e s e n t e d to the p r o g r a m minpro&.
TABLE
O u t p u t o f t h e p r o g r a m minpmbe u s i n g t h e D P B 1-specific oligonucleorides f r o m r e f e r e n c e 14
1
The folhnwing hererozygtms combinarmns cannot he distinguished: 8 genoD'pes in .i groups with up to 2 identical combinations + -
--',-
+
-
-
-
+ -
-
+ . . . . "~~ ++ +- - - + - ÷ - + +- + - - - -~- + - + + - + .... + + - + + .... + .... + +- + + +--++ ~ + .... + F- - + - -
+ +
DPBl'04fll
++ ++ ++ ++ F ~+4 ++
DPBI'02111 DPB i *0202 DPBi "{bif I DPP~| "0402 DPBI'O21II DPBI'02UI DPBI "021)2
DPBI'0801 DPIBl~070! DPBP070! DPBI" 1901 DPB 1" 1901 DPBI'050t DPBI~IgOt DPBI~0801
24
C.R. Miiller and T. H. Eiermann
TABLE 2
Reaction patterns of an optimized set of SSO defining D P B I genotypes $SO
Allele
O1
DPBI'OIOI
+
DPBI'0201
-
DPB
i'0202
.
DPBIb
-
DPB
1 * 0401
.
DPB
1 *0402
-
DPB
I "0501
DPB
1 ~0701
DPB
.
.
.
.
.
.
-
DPBI*1101
.
D P B I * I 4 0 1
.
DPBI'ISOI
+
D P B [ ' 1 6 0 1
-
DPB['I70[
.
DPBI'Ig0i
180l
.
. .
.
-
+
+
.
+
+
.
+
+
+
-
+
+
+
+
.
+
+
+
+
.
-
-
-
+
-
-
+
-
+
.
+
+
-
_
.
. + .
. +
. -
.
.
.
•
-
. +
We found that all informatiun accessible by this method could already be gained using a subset of only 18 SSO. The reaction patterns o f these probes are shown in Table 2. Since many o f the reaction patterns can be obtained by more than one probe, we are curremly investigating which o f these probes shows the best handling under laboratory conditions. For example, it is possible to choose a probe of length 18 for each of the reaction patterns listed in Table 2. At first glance, this result might seem surprising since it shows that the number of SSO probes can be fewer than the number of alleles to be defined. It also means that selecting allele-specific oligonucleotide (ASO) probes, as originally suggested [18], is not the optimal strategy even when possible. Moreover, it is not always possible to find probes specific for each allele, since it is known that the polymorphism of class II genes is in part a patchwork structure of sequence differences generated by gene conversion [19-21]. Reducing the number of probes will be essential for effective work in tissue typing laboratories. For that purpose selecting SSO reacting with a number of alleles is preferable, since the maximum information for typing (usually heterozygous) cells is contained in a probe reacting with about (a + I/2) • (1 - 1/V'2) alleles out of a set of a alleles. Of course, in most cases the smallest defining subset of probes is far from being unique. An exhaustive
.
+
.
+
-
.
.
.
.
.
.
.
. -
+
.
-
-
-
. -
+
-
-
+
-
-
-
+
+
+
-
+
+
+
-
+
+
+
+
+
+
+
+
-
-
q-
-
+
-
+
+
+
+
-
+
+
+
-
-
-
.
+
-
-
÷
. -
+
+
-
-
-
+
-
+
-
-
-
+
+
+
+
.
-
-
. +
.
+
-
.
.
.
-
.
.
.
-
+
.
.
-
.
-
. +
+
.
.
.
+
.
.
.
.
.
+
.
+ .
.
-
+
.
+ .
.
.
.
.
+
. -
.
.
.
.
.
-
+
+ .
.
.
+
18
+
. .
-
-
÷
.
+
-
17
+
-v
-
.
16
.
+
.
+
+
+
+
.
+
+
-
.
.
-
+
.
+
+
+
-
.
-
+ -
-
.
-
-
+
.
+
+
.
.
÷
-
+
.
15
+
.
.
14
+
-r
.
13
-
+
+
.
-
12
.
.
.
.
.
.
.
11
-
-
.
+
-
-
+
+
"~
.
.
+
+ .
+
.
.
.
l0
.
-
+
DPBI°I30I
+
.
.
.
.
-
.
.
. -
1 "0901
DPBI"
.
07
-
.
+
.
09
. .
-
.
DPBI'I201
+
. -
08
. +
.
+
.
06
+
. -
-
. +
.
.
.
05
.
-
.
-
DPBIOl0Ol
04
.
.
DPB|'0801
03
-
.
DPBI'0301
DPBI*0601
G2
.
. -
.
.
+
search for the minimum means traversing the "tree of subsets" o f probes down to each point where a loss of information occurs. Let a denote the number of alleles, p the number of probes, and f ( 0 < f < 1) the fraction of probes that can be omitted, then the computing time cost for a procedure like that can be estimated by the expression l f~2I- I,-v; P(p, a ) 2 P ~ J-v; e-I~-'I~dx where P(p, ~) is some function dominated by a polynomial of order 4 in a and I in p and therefn re nor contributing significantly to the tremendous growth of this expression. However, assuming the other variables constant, the rest o f the expression shows an exponential behaviour with respect to p. For small values o f f , an even more dramatic growth with respect to the number of redundant probes is found. Therefore, with increasing size and redundancy of the set of probes the search can become extremely time consuming. In sets of probes, such as the one examined here, we cannot be sure that the number of probes at the time we stopped our search is definitely the minimum. Our computer simulation revealed another interesting fact. There are two pairs of hererozygous genotypes which are not separable even using all possible SSO (Table 3). Again, as observed before (Table 1), ceils possessing the DPBI~0401, D P B I ' 0 8 1 0 phenotype
HLA-DPB1
TABLE 3
Typing
with SSO
25
DPB[ second exons of hererozygous alielic combinations showing identical hybridization pa~terns with the set of probes from Table 2
DBPI*080t DPB 1*0401
............................
DPB i *0201
..................................................................
DPI3 ! "0701
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A .................
.
.
_
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
AA . . . . . . . . . .
K ......
M.......
GGP~
M ....... .
.
.
.
.
.
.
.
.
.
K
.AA . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
GGP~ .
.
.
.
.
DBPI'0801 DPBI'I701
VH.L ........................................
D ................
VFI,L ...........................................
D .............................
~ ...........
DPB1"1601 D P B 1" 0 9 0 [
OniF pol~'mot~hie nesidues (amino ~cids no. 8 - 8 7 ) different from D P B I ' 0 8 0 1 ar~ ~hnwn T h e braces marked with ° denote regions where crosslng-o,~er could here r=ken place. braces marked with T denote regions where specific primers ¢an be chosen m distinguish these ge~ozy~es.
showed an identical hybridization pattern to DPBI*020 I, DPB l*0701-posifive cells. In contrast, by additional SSO, the other three pairs of nonseparable variants observed previously (Table 1) could be differentiated. Inclusion of recently described alleles (DPvclb, DPBI*t20[, -DPBI*[801) reveated an additional pair of phenotypes (DPB 1"090 l, DPB 1=1601 and DPB ! "0801, DPB 1~t 701; Table 3) with identical hybridization patterns even with the optimized set of SSO. Table 3 illustrates the reason why these situations occur, In both cases one pair of alleles (e.g., DPBI~0701 and DPBI*020I) can be generated by reciprocal crossing-over between the other pair of alleles (e.g., DPBI~0401 and DPBI~0801). Only SSO probes taken from the crossing-over area would solve the problem. But since the crossing-over area spans 24 bp and 60 bp of DNA with two mismatches at the end of the probes, suitable hybridization conditions are unlikely to be attained [22]. Therefore, some individuals cannot be definitely typed by simple genotyping. This may be relevant for typing of donor pools for unrelated bone marrow transplantation, where family typing seems rather impractical to resolve ambiguous phenotypes. Alternative methods to resolve such ambiguous phenorypes ~tould be to perform a DNA amplification using primers chosen from the polymorphic ends of the DPB[ second exon (Table 3) [23]. This would allow amplification of only one of the two alleles involved in each phenotype. Then these alleles can be distinguished by hybridization with a single probe. Extending this method could even resolve double crossing-over situations involving three genes with large homologous parts between the polymorphisms enclosing the crossing-
over sites. Currently this still appears to be less work than sequencing the amplified DNA of the cell. CONCLUSIONS In conclusion we have shown that computer simulation is a valuable tool to optimize sets of 550 for HLA-DP typing, it has been ~ble to detect previously unknown equivocal patterns of hybridization by variant heterozygous phenotypes in a recendy published series of oiigonucleotides. We were ~bie to define an optimized s e t of oligonucleotides which allows the definition of 72 alleles using only 18 different SSO. Testing the vM~e of these probes in practical use is unde¢ way ~nd hybridization and washing conditions of these probes w i l l be detailed elsewhere~ Moreover minprobeshowed the limkalion of this new genoryping method given by the f~ct that some alleles may be the result of reciprocal crossing-over events generating phenotypes not separable by simple genotyping of DPB1 genes.
ACKNOWLE'DGM ~NT$
We thank M. Schimmer for writing our DNA editor and m a i n t a i n i n g o u r DNA database and !. Rich for critk~ reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschaft SFB 322 C5. REFERENCES 1. D a v i s M M ,
Btorkman
PJ: T-ceil dntigen
receptor
genes
and T-ceU recognition. Nature 334:395, 1988. 2. Brown JH, J~de~zky T, Saper MA, Samr=oul 8, Bjorkman PJ, Wiles CD: A hypothetical mode[ of the
26
foreign antigen binding site of class 11 histocompatibility molecules. Nature 322:845, 1988. 3. Odum N, Pla:z P, Jakobsen BK, Petersen CM, Jacobsen N, Moiler J. Ryder LP, Lamm L, Sveigaard A: HLA-DP and bone marrow transplantation: DP-incompatibility and severe acute graft versus host disease. Tissue Antigens 30:213, 1987. 4. Amar A, Nepom GT, Mickelson EM, Erlich HA, I lansen JA: HLA-DP and HLA-DO genes in presumptive HLAidentical siblings: Structural and functional identification of altelic variation. J Immunol 138:1947, 1987. 5. Shaw S, Johnson AH, Shearer GM: Evidence for a new segregant series of B-cell antigens that are encodcM in the HLA-D region and that stimulate secondary allogeneic proliferative and cytotoxic responses. J Exp Med 152:565, 1980. 6. Termiitelen A, Bradley BA, van Rood Jj: A new determinant defined by PLT, coded for in the HLA region and apparently independent of the HLA-D and DR loci. Tissue Antigens 15:267, 1980. 7_ Tanigaki N, Tosi RM, Parodi B, Snrrentino R, Ferrara GB, Strominger JL: Detection of HLA-DP serological allodeterminants by the use of radio-iodinated DP molecules. Eur J lmmunol 17:743, 1987. 8. Mueller-Eckhardt G, Kiefel V, Schmidt A, Tlusty A, Santoso S, MueUer~Eckhardt C: Discrimination of antibodies against angitens of different MHC loci in human sera by monodonal antibody-specific immobilization of leukocyte antigens. Hum Immunol 25:125, 1989. 9. Bodmer JG, Bodmer WF, Heyes J, So AK, Tonks S, Trowsdale J. JongJ: Identification of HLA-DP polymorphism with DP a and DP # probes and monoclonal antibodies: Correlation with primed lymphocyte typing, Proc Nad Acad Sci USA 84:45r'6, 1987. 10. Hyldig-Nielson JJ, Morling N, Odum N, Ryder LP, Platz P, Jakobsen B, Svejgaard A: Restriction fragment length polymorphism of rhe HLA-DP subregion and correlation of HLA-DP phenotypes. Proc Natl Acad Sci USA 84:1644, 1987. I 1. Maeda M, Inoko H, Andn A, Uryu N, Nagata Y, Tsuji K: HLA-DP typing by analysis of DNA restriction fragment length polymorphisms in the HLA-DP/3 subregion, Hum lmmunol 21:239, 1988. 12. Bugawan TL, Horn GT, Long CM, Mickelson EM, Hunsen JA, Ferrara GB, Angelini G, Erlich HA: Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP-a and DP-fl loci. J lmmunol 1,~h4024, 1988.
C.R. Miilfer and T. 1-t. Eiermann
13. Bugawan TL, Angelini G, Larrick J, Auricchio S, Ferrara GB, Erlich HA: A combination of a particular HLA-DP's allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease. Nature 339:470, 1989. 14. Angelini G, Bugawan TL, Delfino L, Erlich HA, Ferrara GB: HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides. Hu.~ lmmunol 26:169, 1989. 15. Lee JS, Sarmrius S, Briat.~ P, Choi E, Cullen C, Lepaslier D, Yunis EJ: Sequence polymorphism of HLA-DP fl chains. Immunogenetics 29:346, 1989. 16. 8egnvich AB, BugawauTL, Nepom BS, Klitz W, Nepom GT, Erlich HA" A specific HLA-DP fl allele is associated with pauciarticular rheumarhoJd arthritis but not adult rheumathoid arthritis. Proc Natl Acad Sci USA 86:9489, 1989. 17. Gustafsson K, Widmark E, Jonsson A-K, Servenius B, Sachs DH, Larhamm~r D, Rask L, Peterson PA: Class 11 genes of the human maior histocompatibilky complex-evolution of the DP region as deduced from nucleotide sequences of the four genes. J Biol Chem 262:8778, 1987. 18. Salki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA: Analysis of enzymatically amplified fl-globin and HLA DQ a DNA with allele specific probes. Nature 32:163, 1986. 19. Angelini G, De Preval C, GorskiJ, Math B: High resolution analysis of the human HLA-DR potymorphism by hybridization with sequence-specific oligonucleotide probes. Proc Narl Acad Sci USA 83:4489, 1986. 20. Gorski J, Math B: Polymorphism of human ia antigens: Gene conversion between two DR's loci results in a new HLA-D/DR specificity. Nature 322:67, 1986. 21. Tiercy J-M, Gorski J, Betuel H, Freidel AC, Gebuhrer L, jeannet M, Math B: DNA typing of DRw6 subtypes: Correlation with DRB1 and DRB3 allelic sequences by hybridization with oligonucleotide probes. Hum Immunol 24:l, 1989. 22. lkuta S, Tagaki K, Wallace RB, hakura K: Dissociation kinetics of 19 base paired olignnucleotide-DNA duplexes containing different single mismatched base pairs, Nucleic Acids Res 15:797, 1987. 23. Shaft S, Saiki R, Erlich H: New Methodology fo~ HLA class I1 oligoaucleotide typing using polymerase chain reaction (PCR) amplification. Hum lmmunol 23:143, 1988.
