Subgrouping of DR4 Alleles by DNA Heteroduplex Analysis Rosa Sorrentino, Isabella Cascino, and Roberto Tosi
ABSTRACT: Amplified DNA molecules from six DR4 alleles at the DRB1 locus were denatured and cross-hybridized pairwise. Several of the DNA heteroduplexes thus generated were found to possess distinct mobilities in polyacrylamide gel electrophoresis. The degree of retardation as compared to homoduplexes depends strongly on the position of mismatched nucleotide pairs. In critical positions, the type of mispairing also influences the number of heteroduplex bands since a transversion-type substitution yields two reciprocal pairings (purine-purine or pyrimidine-pyrimidine) whereas a transition-type substitution generates symmetrical (purine-pyrimidine) pairings, Based on their heteroduplex pattern the DR4 alleles can be subdivided in four groups: group I = DRB*0401, group II = 02 and 06, group III - 03 and 04, and group IV = 05. Each group can be recognized by the heteroduplex bands generated
by cross-hybridizing with group II reference DNA (either the 02 or 06). This subgrouping is obtained with a single electrophoretic run and without the use of probes. However, the alleles within groups II and III, and notably the alleles 03 and 04, which are both present in the Caucasoid population, can be distinguished only by oligonucleotide hybridization (dot blot) analysis. With this limitation, the method can be recommended either in conjunction with dot blot typing or independently, thus avoiding completely the use of probes in the cases where it is not essential to discriminate between 03 and 04. The data also show that distinguishable heteroduplexes may be generated by a single mismatch. This opens the possibility of applying the same technique to genetic systems of lower degree of polymorphism. Human Immuno/og3 33, 18-23 (1992)
ABBREVIATIONS
PCR
polymerase chain reaction
pur pyr
purine pyrimidine
INTRODUCTION The availability of the polymerase chain reaction (PCR) technique has permitted an analysis of the "micropolymorphism" of H L A allelic sequences and the discovery of many variants which do not give rise to serologically or cellularly definable epitopes [1]. So far, the analysis of these variants has been based on hybridization of the amplified genes with a variety of oligonucleotide probes corresponding to known sequences. The ever increasing n u m b e r of newly described alleles, particularly of the H L A - D R B 1 locus, necessitates the use of a correspondingly large number of oligonucleotide probes. D u e to the patchwork assortFrom the Dipartimento di Biologia Cellulare e dello Sviluppo. Universit,~ "La Sapienza" (R.S.) and Istituto di Biologia Cellulare, CNR (I.C.; R.T.), Roma, Italy. Address reprint requests to Roberto Tosi, Institute of Cell Biology, Viale Marx 43, 00187 Rome, Italy. ReceivedJuly 24, 1991; accepted September 20, 1991.
18 0198-8859/92/$5.00
ment of polymorphic sequences, the presence of a given allele is frequently derived from a pattern of hybridization signals rather than from a single positivity [2]. This poses problems of interpretation, particularly in heterozygous combinations. The present work introduces a simple method of DR4 subgrouping by D N A heteroduplex analysis. This is based on the assumption that D N A heteroduplexes formed by denaturation and renaturation after mixing two alleles will have a double helical structure mismatched in the polymorphic positions, that these mismatches will alter the conformation of the D N A molecule, and that their migration in polyacrylamide gel electrophoresis will be modified. This was found to be true for the D R B 3 gene and the possibility of discriminating DRB3*0101, "0201, and "0301 [3] has been previously demonstrated. The present work applies the same assumptions to Human Immunology 33, 18-23 (1992} © American Society for Histocompatibility and lmmunogenetics, 1992
DR4 DNA Heteroduplexes
the DRB 1 locus. Due to the large number of alleles at this locus, we chose to restrict the analysis to the DR4 subgroup* since the DR4 alleles offer the advantage of being specifically amplifiable [4, 5] and, in addition, they cannot be discriminated by standard cytotoxicity. M A T E R I A L S A N D METHODS
Cell lines. The following cell lines were used: WT51 (DRBI*0401;Dw4), FS(DRBI*0402;Dwl0), SSTO (DRB 1~0403 ;Dw 13), LS40 (DRB 1 *0404 ;Dw 14), KT3 (DRB 1"0405 ;Dw 15), and KT2(DRB 1*0406;DwKT2) [6-8]. Gene amplification. The method described by Saiki et al. [9] was followed. Briefly, 1 /xg of genomic D N A was amplified for 30 cycles using the thermostable D N A polymerase from Thermus aquaticus (Cetus, Emeryville, CA) under the following conditions: reaction volume = 50/xl; MgCl2 concentration = 1.5 mM; cycles = 60 seconds 94°C, 30 seconds 56°C, and 30 seconds 72°C. The oligonucleotide primers were prepared with a Bio-Search Ciclone D N A synthesizer (New Brunswick Scientific Co., San Raphael, CA). Their sequences are as follows: 5 ' - G G A G G A T C C G A G C A G G T T A A A C A T G A G T G T - 3 ' corresponding to nucleotide positions 25 to 45 of D R B I * 0 4 and 5 ' - G A C C C G G G C G A C G T G A C A C T T C G A - 3 ' complementary to positions 279-262 of all DRB alleles. This primer combination specifically amplifies the DRB 1"04 alleles. When labeled amplified D N A was needed, the 5' primer (5 pmol) was end labeled by polynucleotide kinase and 1 pmol was added to the amplification mixture containing 25 pmol of each of the unlabeled primers. The amplified products were 5' end labeled in the coding strands with a specific activity of approximately 5 x 10 6 cpm//zg.
19
Fig. 1. Two more recently described variants, D R B I * 0 4 0 7 and *0408 [5, 10], are identical to *0403 and *0404, respectively, except for the interchange of G and T at positions 257 and 258 and they can only be distinguished by specific oligonucleotide hybridization of this site. Amplification using a DR4-specific 5' primer yielded a D N A fragment of 270 base pairs. The amplification products of cells homozygous for each allele were directly run in polyacrylamide gel electrophoresis. Each allele gave a single (homoduplex) band in the same position. When the alleles were combined pairwise, denatured at 95°C, and renatured at 37°C, the electrophoretic patterns shown in Fig. 2 were obtained. Some of the allele combinations produced either one or two extra bands with distinguishable degree of retardation, as schematized in Fig. 3. To test whether these extra bands corresponded to D N A heteroduplexes and to identify the strand composition of each band, the experiment was repeated after labeling only one o f the four D N A strands involved in each cross-hybridization. This was done by using 32p_
FIGURE 1 DRB*04 allele sequences. Upper part: consensus sequence and primer position of the amplified products. Lower part: nucleotide substitutions at the polymorphic positions in the different DRBI*04 alleles. AMPLIFIED DR4 SEQUENCE PRIMER RESTRSITE 10
20
3O
4O
50
60
25
35
45
55
65
75
GGAGGATCCG AGCAGGTTAA ACATGAGTGT CATTTCTTCA ACGGGACGGA GCGGGTGCGG 70
GAGTACCGGG
GAC*TCCTGG
CGGTGACGGA
GCTGGGGCGG
GGCCG*GGTG
260 275
* The designations of DR4 alleles are generally abbreviated, e.g., DRBI*0401 = 01.
170 185 AGTACTGGAA
22o 235 GACACCTACT
135 CGACGTGGGG 180 195 CAGCCAGAAG
230 245 GCAGACACAA
24o 255 CTACGGGGTT
270 279
TCACAGTGCA
GCGGGCCCAG RESTR SrTE
PRIMER
POLYMORPHIC POSITIONS
RESULTS Six DR4 alleles, D R B l * 0 4 0 1 - 0 6 , corresponding to separate Dw (T-cell recognized) specificities (Dw4, wl0, w13, w14, w15, KT2) were analyzed in the present study. Their sequence variations are shown in
CCT***GCCG
120
125 GCTTCGACAG
160 175
210 225
A**A***GCG
110
115 GAGT*CGTGC
150 165
2o0 215
250 265
100
105 TCACCAAGAG
140 155
190 205
G**GAGAGCT
90
95 GATACTTCTA
130 t45
Polyacrylamide gel electrophoresis. Five-microliter aliquots (out of 50 ¢xl total reaction mixture) o f each amplified D N A were mixed in pairs, boiled for 2 minutes, cooled at room temperature for 10 minutes, and run overnight at room temperature, 10 mA, on a 12% polyacrylamide gel. The latter was stained by ethidium bromide.
80
85 TTCCTGGACA
ALLELES I10
DRBI*0401 02 03 04 05 06
A A A A A C
169 170 171
G G G G A G
A A A A G A
T T T T C T
199
C A C C C C
207 208
G A G G G G
C G C C C C
210 211 212
G C G G G G
A G A A A A
A A G G G G
221
257
258
C C A C C A
G T T T G T
T G 6 G T G
20
R. Sorrentino et al.
D C
G-
A
O
L 01 + 05
01 + 06
02 + 03
02 + 05
03 + 05
01 + 02
03 + 06
05 + 06
04 + 05
04 + 06 02 + 04
FIGURE 3 Schematic representation of the heteroduplex migration patterns. The data shown in Fig. 2 are grouped according to the different distinguishable patterns and the corresponding allele combinations.
Based on the above data, every heteroduplex combination was assigned to a band and its relationship with number, position, and type of mismatches was defined, as schematized in Fig. 5. FIGURE 2 Polyacrylamide gel electrophoresis of PCR-amplified DRBI*04 alleles after pairwise hybridizations. From left: lane 1, alleles "0401+'0403; lane 2, "0401+'0404; lane 3, *0403+*0404; lane 4, *0402+*0406; lane 5, "0401+'0405; lane 6, *0403+*0405; lane 7, *0404+*0405; lane 8, "0401+'0402; lane 9, "0401+'0406; lane 10, *0402+*0403; lane 11, *0403+0406; lane 12, *0402+*0404; lane 13, *0404+0406; lane 14, *0402+*0405; lane 15, *0405 +*0406. Apparent doubling of the homoduplex band in some combinations is due to a comigrating contaminant in the KT2 cell line (*0406 allele). The faint line on top is due to a PCR contaminant in all samples.
labeled 5' primer in the amplification step, thus labeling the coding strand of each allele. Each band was identified unequivocally as possessing the coding strand of one allele only. For instance, band B in the 0 1 + 0 2 combination (Fig. 4, lane 6) possessed the coding strand of the 01 allele, whereas band C possessed the coding strand of the 02 allele. Therefore, band B was identified as the 01/02 heteroduplex, whereas band C was the 02/ 01 heteroduplex (the coding strand is always listed as the first). Similarly, bands D, E, F, and G were identified in the different combinations shown in Fig. 4. The only exception was band A, which contained both 01 and 05 coding strands in the 0 1 + 0 5 hybridization. Therefore, this band is interpreted as being contributed by both 01/05 and 05/01 heteroduplexes.
DISCUSSION The observed electrophoretic behavior of the different DR4 heteroduplexes is obviously related to some structural modification produced by mismatched base pairings. The modification of the superhelical configuration (DNA bending) rather than the conformation of the double helical structure is thought to be the factor affecting migration [11]. The rate of movement through the gel pores depends on the superhelical diameter, which increases with the curvature of the DNA. D N A bending has been found to correlate to specific sequences [ 12] and theoretical models of the relationship between D N A sequences and curvature have been proposed [11, 13]. However, models taking into account not only Watson-Crick pairs but also mismatched pairs are not available. Therefore the "rules" for interpreting the migration of mismatched heteroduplexes must be derived empirically. They may differ from one D N A molecule to another according to length, sequence, number, and position of mismatches. In Fig. 5 the data have been organized in order to visualize such relationships. The bands corresponding to the different heteroduplexes have been listed in order of decreasing migration (or increasing mismatch effect). The mismatches are schematically represented so that their position and their type [purine (pur)-pyrimidine (pyr), pur-pur, pyr-pyr)] are shown at the same
DR4 DNA Heteroduplexes
1
2l
2
3
4
5
6
7
8
9
lg
11
lZ
]3
14
FIGURE 4 Autoradiogram of polyacrylamide gel electrophoresis showing ~2P-labeled coding strands. Lanes 1 and 2: *0401 +'0405 combination where either the DRB 1"0401 (lane 1) or the *0405 (lane 2) coding strand was labeled. Lanes 3 and 4:"0406+0401 combination where either *0406 (lane 3) or "0401 (lane 4) coding strands were labeled. Lanes 5 and 6: the same as for lanes 3 and 4 but *0406 was replaced by *0402. Lanes 7 and 8:*0402+*0403 combination where either *0402 (lane 7) or *0403 (lane 8) coding strand was labeled. Lanes 9 and 10: the same as for lanes 7 and 8 but *0402 was replaced by *0406. Lanes 11 and 12:*0405 +*0402 combination where either *0405 (lane 11) or *0402 (lane 12) coding strand was labeled. Lanes 13 and 14: same as for lanes 11 and 12 but *0402 was replaced by *0406. FIGURE 5 Schematic representation of heteroduplex mispairings and assignment of each band to the corresponding heteroduplexes. For each allele combination, the coding strand is on the left. The mismatches in the heteroduplex pairs are shown schematically as white circles (pur-pyr), shaded circles (pur-pur), or black circles (pyr-pyr). The assignment of a band to the corresponding group of heteroduplexes is based on the results shown in Figs. 2 and 4. i i 0 169 170 171 DNA
,,~,~.0~.~
199
Iolo o ololo
207 208
2 1 0 21 ~ 2 1 2 2 2 1 2-~7 2 5 B
olo
o olOlO
05/0,
0 @ • @ 0 @ • 0 • @
o~/o4
04/c I *s/o4
O
U4/03
•
~2/o6 06102 ~/os
oZl . ^ . o
@ •
05/0 ! 03/05 05/03 04/05 05/04 02/03 02104
@ 0 @ 0 0 0 • • 0 • @0 0 @ 0 0 0 0 000 0 000 @0@ 000 0@0 000 0@ O00 @0 @O®OOO0 @0@000
06/03 06/04
o~/o~ o6,'os o2eo~ o~/ol
• •
•
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03106
0
04Jo~ 04/02 0~,'0":
@
011c3b
@
,5,'o~ s,'o,
0 0 0 • ® 0 Q Q
0 •
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@ @ @
•
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@0 0 ® •
•
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@• @•
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B
time. The complexity of the system can be immediately perceived. H o w e v e r , a rationale for the observed data can be outlined as follows: 1. A mismatch in position 110 has a marked effect on electrophoretic mobility. This is evidently seen in the 0 3 + 0 6 combination, where the two alleles differ only in this position. Since this is a transversion-type substitution, the two reciprocal heteroduplexes are different, possessing either a pur-pur or a pyr-pyr mismatch. The two corresponding bands, E and D, were identified by labeling the coding strand (Fig. 4) and the slower migrating, D, was that possessing the pur-pur pairing. 2. A group of six mismatches ( 1 9 9 - 2 1 2 ) has the same retardation effect as the single mismatch in position 110. (02+04, 02+03). 3. The three consecutive mismatches at positions 1 6 9 171 generate a single retarded band (01+05, 0 3 + 0 5 , 04+05). This was expected since all three substitutions are of the transition-type and thus give rise to two symmetrical heteroduplexes, both with pur-pyr pairings. 4. T h e mismatches in the 3' end of the molecule, from 212 onwards, have no influence on retardation by themselves (01+03, 0 1 + 0 4 , 0 3 + 0 4 ) and little influence when combined with other substitutions ( 0 1 + 0 6 versus 03+06). 5. Usually, the retardation effects of the different mismatches are additive. An exception is the 0 2 + 0 6 combination where they neutralize each other. Apparently, the above "rules" cannot be generalized to other D N A fragments. Heteroduplexes between DRB3 locus alleles have been previously analyzed [3] and, in that case, the differences in migration depended
22
mainly on the number of mismatches regardless of their positions and on the type of mispairing (pur-pyr, purpur, or pyr-pyr), the heteroduplex possessing more pyrpyr pairings being always the slower migrating. The only common rule so far is that a mismatch that has a retardation effect will generate two bands if the substitution is of the transversion type, one band if it is of the transition type. These empirical data may be possibly accommodated in a nearest-neighbor conformational model [14] by suitably modifying rotation matrices. Only then will it be possible to make predictions on the migration of mismatched heteroduplexes in the different systems. The most important corollary of these data is that a single mismatch can cause a marked electrophoretic retardation. This demonstrates the possibility of detecting new minor subtypes by this method, even if they involve a single substitution, provided that it occurs at a critical site and, more important, it shows that it can be utilized to reveal polymorphism also in systems other than the major histocompatibility complex, where multiple substitutions are not expected. In general, this method can be proposed as a first, simple tool for detection of genomic variability. Next, the practical applicability of this method to DR4 typing must be considered. Based on the observed heteroduplex bands (Fig. 3), the DR4 alleles can be subdivided in four groups (Table 1). Each group can be distinguished from the others based on the heteroduplex bands generated in pairwise combinations. In practice, a D N A sample can be attributed to one of the four groups by mixing it to "group II" D N A (either the 02 or the 06 amplified allele). The heteroduplex bands thus generated will be different if the unknown sample is 01 (bands B + C ) , 03 or 04 (bands D+E), or 05 (bands F+G). N o heteroduplex band will be seen if the unknown sample is 02 or 06. Clearly, while 01 and 05 can be typed unequivocally as individual alleles, 02 cannot be discriminated from 06 and 03 cannot be discriminated from 04. Practically, for the typing of Caucasoid populations, the lack of discrimination between 02 and 06 is marginally important because 06 is almost exclusively observed in Oriental populations [16]. Instead, 03 and 04 occur in the same population and they need to be distinguished from each other. At present, this can only be done, apart from cellular typing, by hybridizing with an oligonucleotide probe centered on position 221, i.e., the only nucleotide substitution between the two alleles (Fig. 1). Consequently, the present method cannot entirely substitute dot blot analysis if one needs to discriminate between the two above alleles. The same limitations apply to matching for transplants. DR4-amplified D N A from donor and recipient will give heteroduplex bands when mixed, if they possess different
R. Sorrentino et al.
TABLE 1
DR4 subgrouping according to heteroduplexes Heteroduplexes formed with s u b g r o u p
Subgroup
D R 4 allele
I
II
III
IV
I
01
--
B+C
--
A
II
02, 06
B+C
--
D+E
F+G
III
03, 04
--
D+E
--
A
IV
05
A
F+G
A
--
DR4 alleles. Again, a 03 versus 04 mismatch will not be detected. Nevertheless, the present procedure is so simple and convenient that, even with the above limitations, it can be used in conjunction with dot blot typing, with the advantage of reducing the number of probes required to a single one and of providing an independent validation of dot blot results. It may be used as the only method in cases where it is not essential to discriminate between 03 and 04.
ACKNOWLEDGMENTS
The DR4 5'-specific primer was a gift of Dr. Giulio Ratti. The technical help of Ms. Eleuteria Lancia and Ms. Irene Pauselli is gratefully acknowledged. This work has been supported by Progetto Finalizzato CNR Biotecnologia e Biostrumentazione and by Progetto Finalizzato CNR Ingegneria Genetica. REFERENCES 1. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA: Analysis of enzymatically amplified beta-globin and HLADQ alpha DNA with allele-specific oligonucleotide probes. Nature 324:163, 1986. 2. Marsh SGE, Bodmer JG: HLA-DRB nucleotide sequences 1990. Immunogenetics 31:141, 1990. 3. Sorrentino R, Iannicola C, Costanzi S, Chersi A, Tosi R: Detection of complex alleles by direct analysis of DNA heteroduplexes. Immunogenetics 33:118, 1991. 4. Gao X, Fernandez-Vina M, Shumway W, Stasmy P: DNA typing for class II HLA antigens with allele-specific or group-specific amplification. I. Typing for subsets of HLA-DR4. Hum Immunol 27:40, 1990. 5. LanchburyJSS, Hall MA, Wesh KI, Panayi GS: Sequence analysis of HLA-DR4B1 subtypes: Additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing. Hum Immunol 27:136, 1990. 6. Gregensen PK, Shen M, Song QL, Merryman P, Degar S, Seki T, Maccari J, Goldberg D, Murphy H, Schwenzer J, Wang CY, Winchester R, Nepom GT, Silver J: Molecular
DR4 DNA Heteroduplexes
23
diversity of HLA-DR4 haplotypes. Proc Natl Acad Sci USA 83:2642, 1986.
1 I. Calladine CR, Drew HR, McCall MJ: The intrinsic curvature of DNA in solution. J Mol Biol 201:127, 1988.
7. CairnsJS, CurtsingerJM, DaM CA, Freeman S, Alter BJ, Bach FH: Sequence polymorphism of HLA-DRB 1 alleles relating to T-cell recognized determinants. Nature 317:166, 1985.
12. Koo HS, Wu HM, Crothers DM: DNA bending at adenine thymine tracts. Nature 320:501, 1986.
8. Gregersen PK, Goyert SM, Song QL, Silver J: Microheterogeneity of HLA-DR4 haplotypes: DNA sequence analysis of LD "KT2" and LD "TAS" haplotypes. Hum Immunol 19:287, 1987. 9. Saiki RK,~Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Herlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Sciente 239:487, 1988. 10. Gregersen PK, Todd JA, Erlich HA, Long E, Servenius B, Choi E, Kao HT, LeeJS: First domain sequence diversity of DR and DQ subregion alleles. In Dupont B (ed): Immunobiology of HLA. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989, p. 1027.
13. De Santis P, Palleschi A, Savino M, Scipioni A: A theoretical model of DNA curvature. Biophys Chem 32:305, 1988. 14. De Santis P, Palleschi A, Savino M, Scipioni A: Validity of the nearest-neighbor approximation in the evaluation of the electrophoretic manifestations of DNA curvature. Biochemistry 29:9269, 1990. 15. Clay TM, Bidwell JL, Howard MR, Bradley BA: PCR fingerprinting for selection of HLA matched unrelated marrow donors. Lancet 337:1049, 1991. 16. Gao X, Fernandez-Vina M, Shumway W, Stasmy P: DNA typing for class II HLA antigens with allele-specific or group-specific amplification. I. Typing for subsets of HLA-DR4. Hum Immunol 27:40, 1990.
ABSTRACT: Amplified DNA molecules from six DR4 alleles at the DRB1 locus were denatured and cross-hybridized pairwise. Several of the DNA heteroduplexes thus generated were found to possess distinct mobilities in polyacrylamide gel electrophoresis. The degree of retardation as compared to homoduplexes depends strongly on the position of mismatched nucleotide pairs. In critical positions, the type of mispairing also influences the number of heteroduplex bands since a transversion-type substitution yields two reciprocal pairings (purine-purine or pyrimidine-pyrimidine) whereas a transition-type substitution generates symmetrical (purine-pyrimidine) pairings, Based on their heteroduplex pattern the DR4 alleles can be subdivided in four groups: group I = DRB*0401, group II = 02 and 06, group III - 03 and 04, and group IV = 05. Each group can be recognized by the heteroduplex bands generated
by cross-hybridizing with group II reference DNA (either the 02 or 06). This subgrouping is obtained with a single electrophoretic run and without the use of probes. However, the alleles within groups II and III, and notably the alleles 03 and 04, which are both present in the Caucasoid population, can be distinguished only by oligonucleotide hybridization (dot blot) analysis. With this limitation, the method can be recommended either in conjunction with dot blot typing or independently, thus avoiding completely the use of probes in the cases where it is not essential to discriminate between 03 and 04. The data also show that distinguishable heteroduplexes may be generated by a single mismatch. This opens the possibility of applying the same technique to genetic systems of lower degree of polymorphism. Human Immuno/og3 33, 18-23 (1992)
ABBREVIATIONS
PCR
polymerase chain reaction
pur pyr
purine pyrimidine
INTRODUCTION The availability of the polymerase chain reaction (PCR) technique has permitted an analysis of the "micropolymorphism" of H L A allelic sequences and the discovery of many variants which do not give rise to serologically or cellularly definable epitopes [1]. So far, the analysis of these variants has been based on hybridization of the amplified genes with a variety of oligonucleotide probes corresponding to known sequences. The ever increasing n u m b e r of newly described alleles, particularly of the H L A - D R B 1 locus, necessitates the use of a correspondingly large number of oligonucleotide probes. D u e to the patchwork assortFrom the Dipartimento di Biologia Cellulare e dello Sviluppo. Universit,~ "La Sapienza" (R.S.) and Istituto di Biologia Cellulare, CNR (I.C.; R.T.), Roma, Italy. Address reprint requests to Roberto Tosi, Institute of Cell Biology, Viale Marx 43, 00187 Rome, Italy. ReceivedJuly 24, 1991; accepted September 20, 1991.
18 0198-8859/92/$5.00
ment of polymorphic sequences, the presence of a given allele is frequently derived from a pattern of hybridization signals rather than from a single positivity [2]. This poses problems of interpretation, particularly in heterozygous combinations. The present work introduces a simple method of DR4 subgrouping by D N A heteroduplex analysis. This is based on the assumption that D N A heteroduplexes formed by denaturation and renaturation after mixing two alleles will have a double helical structure mismatched in the polymorphic positions, that these mismatches will alter the conformation of the D N A molecule, and that their migration in polyacrylamide gel electrophoresis will be modified. This was found to be true for the D R B 3 gene and the possibility of discriminating DRB3*0101, "0201, and "0301 [3] has been previously demonstrated. The present work applies the same assumptions to Human Immunology 33, 18-23 (1992} © American Society for Histocompatibility and lmmunogenetics, 1992
DR4 DNA Heteroduplexes
the DRB 1 locus. Due to the large number of alleles at this locus, we chose to restrict the analysis to the DR4 subgroup* since the DR4 alleles offer the advantage of being specifically amplifiable [4, 5] and, in addition, they cannot be discriminated by standard cytotoxicity. M A T E R I A L S A N D METHODS
Cell lines. The following cell lines were used: WT51 (DRBI*0401;Dw4), FS(DRBI*0402;Dwl0), SSTO (DRB 1~0403 ;Dw 13), LS40 (DRB 1 *0404 ;Dw 14), KT3 (DRB 1"0405 ;Dw 15), and KT2(DRB 1*0406;DwKT2) [6-8]. Gene amplification. The method described by Saiki et al. [9] was followed. Briefly, 1 /xg of genomic D N A was amplified for 30 cycles using the thermostable D N A polymerase from Thermus aquaticus (Cetus, Emeryville, CA) under the following conditions: reaction volume = 50/xl; MgCl2 concentration = 1.5 mM; cycles = 60 seconds 94°C, 30 seconds 56°C, and 30 seconds 72°C. The oligonucleotide primers were prepared with a Bio-Search Ciclone D N A synthesizer (New Brunswick Scientific Co., San Raphael, CA). Their sequences are as follows: 5 ' - G G A G G A T C C G A G C A G G T T A A A C A T G A G T G T - 3 ' corresponding to nucleotide positions 25 to 45 of D R B I * 0 4 and 5 ' - G A C C C G G G C G A C G T G A C A C T T C G A - 3 ' complementary to positions 279-262 of all DRB alleles. This primer combination specifically amplifies the DRB 1"04 alleles. When labeled amplified D N A was needed, the 5' primer (5 pmol) was end labeled by polynucleotide kinase and 1 pmol was added to the amplification mixture containing 25 pmol of each of the unlabeled primers. The amplified products were 5' end labeled in the coding strands with a specific activity of approximately 5 x 10 6 cpm//zg.
19
Fig. 1. Two more recently described variants, D R B I * 0 4 0 7 and *0408 [5, 10], are identical to *0403 and *0404, respectively, except for the interchange of G and T at positions 257 and 258 and they can only be distinguished by specific oligonucleotide hybridization of this site. Amplification using a DR4-specific 5' primer yielded a D N A fragment of 270 base pairs. The amplification products of cells homozygous for each allele were directly run in polyacrylamide gel electrophoresis. Each allele gave a single (homoduplex) band in the same position. When the alleles were combined pairwise, denatured at 95°C, and renatured at 37°C, the electrophoretic patterns shown in Fig. 2 were obtained. Some of the allele combinations produced either one or two extra bands with distinguishable degree of retardation, as schematized in Fig. 3. To test whether these extra bands corresponded to D N A heteroduplexes and to identify the strand composition of each band, the experiment was repeated after labeling only one o f the four D N A strands involved in each cross-hybridization. This was done by using 32p_
FIGURE 1 DRB*04 allele sequences. Upper part: consensus sequence and primer position of the amplified products. Lower part: nucleotide substitutions at the polymorphic positions in the different DRBI*04 alleles. AMPLIFIED DR4 SEQUENCE PRIMER RESTRSITE 10
20
3O
4O
50
60
25
35
45
55
65
75
GGAGGATCCG AGCAGGTTAA ACATGAGTGT CATTTCTTCA ACGGGACGGA GCGGGTGCGG 70
GAGTACCGGG
GAC*TCCTGG
CGGTGACGGA
GCTGGGGCGG
GGCCG*GGTG
260 275
* The designations of DR4 alleles are generally abbreviated, e.g., DRBI*0401 = 01.
170 185 AGTACTGGAA
22o 235 GACACCTACT
135 CGACGTGGGG 180 195 CAGCCAGAAG
230 245 GCAGACACAA
24o 255 CTACGGGGTT
270 279
TCACAGTGCA
GCGGGCCCAG RESTR SrTE
PRIMER
POLYMORPHIC POSITIONS
RESULTS Six DR4 alleles, D R B l * 0 4 0 1 - 0 6 , corresponding to separate Dw (T-cell recognized) specificities (Dw4, wl0, w13, w14, w15, KT2) were analyzed in the present study. Their sequence variations are shown in
CCT***GCCG
120
125 GCTTCGACAG
160 175
210 225
A**A***GCG
110
115 GAGT*CGTGC
150 165
2o0 215
250 265
100
105 TCACCAAGAG
140 155
190 205
G**GAGAGCT
90
95 GATACTTCTA
130 t45
Polyacrylamide gel electrophoresis. Five-microliter aliquots (out of 50 ¢xl total reaction mixture) o f each amplified D N A were mixed in pairs, boiled for 2 minutes, cooled at room temperature for 10 minutes, and run overnight at room temperature, 10 mA, on a 12% polyacrylamide gel. The latter was stained by ethidium bromide.
80
85 TTCCTGGACA
ALLELES I10
DRBI*0401 02 03 04 05 06
A A A A A C
169 170 171
G G G G A G
A A A A G A
T T T T C T
199
C A C C C C
207 208
G A G G G G
C G C C C C
210 211 212
G C G G G G
A G A A A A
A A G G G G
221
257
258
C C A C C A
G T T T G T
T G 6 G T G
20
R. Sorrentino et al.
D C
G-
A
O
L 01 + 05
01 + 06
02 + 03
02 + 05
03 + 05
01 + 02
03 + 06
05 + 06
04 + 05
04 + 06 02 + 04
FIGURE 3 Schematic representation of the heteroduplex migration patterns. The data shown in Fig. 2 are grouped according to the different distinguishable patterns and the corresponding allele combinations.
Based on the above data, every heteroduplex combination was assigned to a band and its relationship with number, position, and type of mismatches was defined, as schematized in Fig. 5. FIGURE 2 Polyacrylamide gel electrophoresis of PCR-amplified DRBI*04 alleles after pairwise hybridizations. From left: lane 1, alleles "0401+'0403; lane 2, "0401+'0404; lane 3, *0403+*0404; lane 4, *0402+*0406; lane 5, "0401+'0405; lane 6, *0403+*0405; lane 7, *0404+*0405; lane 8, "0401+'0402; lane 9, "0401+'0406; lane 10, *0402+*0403; lane 11, *0403+0406; lane 12, *0402+*0404; lane 13, *0404+0406; lane 14, *0402+*0405; lane 15, *0405 +*0406. Apparent doubling of the homoduplex band in some combinations is due to a comigrating contaminant in the KT2 cell line (*0406 allele). The faint line on top is due to a PCR contaminant in all samples.
labeled 5' primer in the amplification step, thus labeling the coding strand of each allele. Each band was identified unequivocally as possessing the coding strand of one allele only. For instance, band B in the 0 1 + 0 2 combination (Fig. 4, lane 6) possessed the coding strand of the 01 allele, whereas band C possessed the coding strand of the 02 allele. Therefore, band B was identified as the 01/02 heteroduplex, whereas band C was the 02/ 01 heteroduplex (the coding strand is always listed as the first). Similarly, bands D, E, F, and G were identified in the different combinations shown in Fig. 4. The only exception was band A, which contained both 01 and 05 coding strands in the 0 1 + 0 5 hybridization. Therefore, this band is interpreted as being contributed by both 01/05 and 05/01 heteroduplexes.
DISCUSSION The observed electrophoretic behavior of the different DR4 heteroduplexes is obviously related to some structural modification produced by mismatched base pairings. The modification of the superhelical configuration (DNA bending) rather than the conformation of the double helical structure is thought to be the factor affecting migration [11]. The rate of movement through the gel pores depends on the superhelical diameter, which increases with the curvature of the DNA. D N A bending has been found to correlate to specific sequences [ 12] and theoretical models of the relationship between D N A sequences and curvature have been proposed [11, 13]. However, models taking into account not only Watson-Crick pairs but also mismatched pairs are not available. Therefore the "rules" for interpreting the migration of mismatched heteroduplexes must be derived empirically. They may differ from one D N A molecule to another according to length, sequence, number, and position of mismatches. In Fig. 5 the data have been organized in order to visualize such relationships. The bands corresponding to the different heteroduplexes have been listed in order of decreasing migration (or increasing mismatch effect). The mismatches are schematically represented so that their position and their type [purine (pur)-pyrimidine (pyr), pur-pur, pyr-pyr)] are shown at the same
DR4 DNA Heteroduplexes
1
2l
2
3
4
5
6
7
8
9
lg
11
lZ
]3
14
FIGURE 4 Autoradiogram of polyacrylamide gel electrophoresis showing ~2P-labeled coding strands. Lanes 1 and 2: *0401 +'0405 combination where either the DRB 1"0401 (lane 1) or the *0405 (lane 2) coding strand was labeled. Lanes 3 and 4:"0406+0401 combination where either *0406 (lane 3) or "0401 (lane 4) coding strands were labeled. Lanes 5 and 6: the same as for lanes 3 and 4 but *0406 was replaced by *0402. Lanes 7 and 8:*0402+*0403 combination where either *0402 (lane 7) or *0403 (lane 8) coding strand was labeled. Lanes 9 and 10: the same as for lanes 7 and 8 but *0402 was replaced by *0406. Lanes 11 and 12:*0405 +*0402 combination where either *0405 (lane 11) or *0402 (lane 12) coding strand was labeled. Lanes 13 and 14: same as for lanes 11 and 12 but *0402 was replaced by *0406. FIGURE 5 Schematic representation of heteroduplex mispairings and assignment of each band to the corresponding heteroduplexes. For each allele combination, the coding strand is on the left. The mismatches in the heteroduplex pairs are shown schematically as white circles (pur-pyr), shaded circles (pur-pur), or black circles (pyr-pyr). The assignment of a band to the corresponding group of heteroduplexes is based on the results shown in Figs. 2 and 4. i i 0 169 170 171 DNA
,,~,~.0~.~
199
Iolo o ololo
207 208
2 1 0 21 ~ 2 1 2 2 2 1 2-~7 2 5 B
olo
o olOlO
05/0,
0 @ • @ 0 @ • 0 • @
o~/o4
04/c I *s/o4
O
U4/03
•
~2/o6 06102 ~/os
oZl . ^ . o
@ •
05/0 ! 03/05 05/03 04/05 05/04 02/03 02104
@ 0 @ 0 0 0 • • 0 • @0 0 @ 0 0 0 0 000 0 000 @0@ 000 0@0 000 0@ O00 @0 @O®OOO0 @0@000
06/03 06/04
o~/o~ o6,'os o2eo~ o~/ol
• •
•
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0
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E
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03106
0
04Jo~ 04/02 0~,'0":
@
011c3b
@
,5,'o~ s,'o,
0 0 0 • ® 0 Q Q
0 •
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@ @ @
•
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time. The complexity of the system can be immediately perceived. H o w e v e r , a rationale for the observed data can be outlined as follows: 1. A mismatch in position 110 has a marked effect on electrophoretic mobility. This is evidently seen in the 0 3 + 0 6 combination, where the two alleles differ only in this position. Since this is a transversion-type substitution, the two reciprocal heteroduplexes are different, possessing either a pur-pur or a pyr-pyr mismatch. The two corresponding bands, E and D, were identified by labeling the coding strand (Fig. 4) and the slower migrating, D, was that possessing the pur-pur pairing. 2. A group of six mismatches ( 1 9 9 - 2 1 2 ) has the same retardation effect as the single mismatch in position 110. (02+04, 02+03). 3. The three consecutive mismatches at positions 1 6 9 171 generate a single retarded band (01+05, 0 3 + 0 5 , 04+05). This was expected since all three substitutions are of the transition-type and thus give rise to two symmetrical heteroduplexes, both with pur-pyr pairings. 4. T h e mismatches in the 3' end of the molecule, from 212 onwards, have no influence on retardation by themselves (01+03, 0 1 + 0 4 , 0 3 + 0 4 ) and little influence when combined with other substitutions ( 0 1 + 0 6 versus 03+06). 5. Usually, the retardation effects of the different mismatches are additive. An exception is the 0 2 + 0 6 combination where they neutralize each other. Apparently, the above "rules" cannot be generalized to other D N A fragments. Heteroduplexes between DRB3 locus alleles have been previously analyzed [3] and, in that case, the differences in migration depended
22
mainly on the number of mismatches regardless of their positions and on the type of mispairing (pur-pyr, purpur, or pyr-pyr), the heteroduplex possessing more pyrpyr pairings being always the slower migrating. The only common rule so far is that a mismatch that has a retardation effect will generate two bands if the substitution is of the transversion type, one band if it is of the transition type. These empirical data may be possibly accommodated in a nearest-neighbor conformational model [14] by suitably modifying rotation matrices. Only then will it be possible to make predictions on the migration of mismatched heteroduplexes in the different systems. The most important corollary of these data is that a single mismatch can cause a marked electrophoretic retardation. This demonstrates the possibility of detecting new minor subtypes by this method, even if they involve a single substitution, provided that it occurs at a critical site and, more important, it shows that it can be utilized to reveal polymorphism also in systems other than the major histocompatibility complex, where multiple substitutions are not expected. In general, this method can be proposed as a first, simple tool for detection of genomic variability. Next, the practical applicability of this method to DR4 typing must be considered. Based on the observed heteroduplex bands (Fig. 3), the DR4 alleles can be subdivided in four groups (Table 1). Each group can be distinguished from the others based on the heteroduplex bands generated in pairwise combinations. In practice, a D N A sample can be attributed to one of the four groups by mixing it to "group II" D N A (either the 02 or the 06 amplified allele). The heteroduplex bands thus generated will be different if the unknown sample is 01 (bands B + C ) , 03 or 04 (bands D+E), or 05 (bands F+G). N o heteroduplex band will be seen if the unknown sample is 02 or 06. Clearly, while 01 and 05 can be typed unequivocally as individual alleles, 02 cannot be discriminated from 06 and 03 cannot be discriminated from 04. Practically, for the typing of Caucasoid populations, the lack of discrimination between 02 and 06 is marginally important because 06 is almost exclusively observed in Oriental populations [16]. Instead, 03 and 04 occur in the same population and they need to be distinguished from each other. At present, this can only be done, apart from cellular typing, by hybridizing with an oligonucleotide probe centered on position 221, i.e., the only nucleotide substitution between the two alleles (Fig. 1). Consequently, the present method cannot entirely substitute dot blot analysis if one needs to discriminate between the two above alleles. The same limitations apply to matching for transplants. DR4-amplified D N A from donor and recipient will give heteroduplex bands when mixed, if they possess different
R. Sorrentino et al.
TABLE 1
DR4 subgrouping according to heteroduplexes Heteroduplexes formed with s u b g r o u p
Subgroup
D R 4 allele
I
II
III
IV
I
01
--
B+C
--
A
II
02, 06
B+C
--
D+E
F+G
III
03, 04
--
D+E
--
A
IV
05
A
F+G
A
--
DR4 alleles. Again, a 03 versus 04 mismatch will not be detected. Nevertheless, the present procedure is so simple and convenient that, even with the above limitations, it can be used in conjunction with dot blot typing, with the advantage of reducing the number of probes required to a single one and of providing an independent validation of dot blot results. It may be used as the only method in cases where it is not essential to discriminate between 03 and 04.
ACKNOWLEDGMENTS
The DR4 5'-specific primer was a gift of Dr. Giulio Ratti. The technical help of Ms. Eleuteria Lancia and Ms. Irene Pauselli is gratefully acknowledged. This work has been supported by Progetto Finalizzato CNR Biotecnologia e Biostrumentazione and by Progetto Finalizzato CNR Ingegneria Genetica. REFERENCES 1. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA: Analysis of enzymatically amplified beta-globin and HLADQ alpha DNA with allele-specific oligonucleotide probes. Nature 324:163, 1986. 2. Marsh SGE, Bodmer JG: HLA-DRB nucleotide sequences 1990. Immunogenetics 31:141, 1990. 3. Sorrentino R, Iannicola C, Costanzi S, Chersi A, Tosi R: Detection of complex alleles by direct analysis of DNA heteroduplexes. Immunogenetics 33:118, 1991. 4. Gao X, Fernandez-Vina M, Shumway W, Stasmy P: DNA typing for class II HLA antigens with allele-specific or group-specific amplification. I. Typing for subsets of HLA-DR4. Hum Immunol 27:40, 1990. 5. LanchburyJSS, Hall MA, Wesh KI, Panayi GS: Sequence analysis of HLA-DR4B1 subtypes: Additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing. Hum Immunol 27:136, 1990. 6. Gregensen PK, Shen M, Song QL, Merryman P, Degar S, Seki T, Maccari J, Goldberg D, Murphy H, Schwenzer J, Wang CY, Winchester R, Nepom GT, Silver J: Molecular
DR4 DNA Heteroduplexes
23
diversity of HLA-DR4 haplotypes. Proc Natl Acad Sci USA 83:2642, 1986.
1 I. Calladine CR, Drew HR, McCall MJ: The intrinsic curvature of DNA in solution. J Mol Biol 201:127, 1988.
7. CairnsJS, CurtsingerJM, DaM CA, Freeman S, Alter BJ, Bach FH: Sequence polymorphism of HLA-DRB 1 alleles relating to T-cell recognized determinants. Nature 317:166, 1985.
12. Koo HS, Wu HM, Crothers DM: DNA bending at adenine thymine tracts. Nature 320:501, 1986.
8. Gregersen PK, Goyert SM, Song QL, Silver J: Microheterogeneity of HLA-DR4 haplotypes: DNA sequence analysis of LD "KT2" and LD "TAS" haplotypes. Hum Immunol 19:287, 1987. 9. Saiki RK,~Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Herlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Sciente 239:487, 1988. 10. Gregersen PK, Todd JA, Erlich HA, Long E, Servenius B, Choi E, Kao HT, LeeJS: First domain sequence diversity of DR and DQ subregion alleles. In Dupont B (ed): Immunobiology of HLA. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989, p. 1027.
13. De Santis P, Palleschi A, Savino M, Scipioni A: A theoretical model of DNA curvature. Biophys Chem 32:305, 1988. 14. De Santis P, Palleschi A, Savino M, Scipioni A: Validity of the nearest-neighbor approximation in the evaluation of the electrophoretic manifestations of DNA curvature. Biochemistry 29:9269, 1990. 15. Clay TM, Bidwell JL, Howard MR, Bradley BA: PCR fingerprinting for selection of HLA matched unrelated marrow donors. Lancet 337:1049, 1991. 16. Gao X, Fernandez-Vina M, Shumway W, Stasmy P: DNA typing for class II HLA antigens with allele-specific or group-specific amplification. I. Typing for subsets of HLA-DR4. Hum Immunol 27:40, 1990.
