Unambiguous typing for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes

Unambiguous Typing for HLA-DQ TA10 and 2B3 Specificities Using Specific Oligonucleotide Probes M. J. H. Kenter, J. D. H. Anholts, G. M. Th. Schreuder, M. C. J. A. van Eggermond, G. M. Ghyselen, J. J. van Rood, and M. J. Giphart

A B S T R A C T : Oligonucleotide probes specificfor the serologically defined TA 10 and 2B3 specificities were

selected based on a comparison of the available HLA-DQ# sequences. Panel and family segregation studies confirm a complete correlation between the reactivities of the selected probes and the TA 10/IIB3 antibodies. The Glu residue at position 45 of the HLA-DQ3 chain is specificfor the TA 10 determinants, and a DQ~ Gly-Val-Tyr sequence is found at position 4 5 - 4 7 for all 2B3positive DQB chains. ABBREVIATIONS

B-LCL MoAb

B-lymphoblastoidcell line monoclonal antibody

PCR SSPE

polymerase chain reaction saline sodium phosphate EDTA

INTRODUCTION The extensive HLA-DQ c~ and/3 polymorphism has been studied by serologic, cellular, biochemical, and molecular biological techniques [1-11]. Using monoclonal antibodies (MoAbs) the DQ molecules can be divided into three groups: (1) IIB3-positive, (2)TA10-positive, and (3) IIB3/TA10-double-negative (predominantly DQw2) DQ molecules [2,9,13-15]. The MoAb TA10 reacts with DQw3.1; MoAb IIB3 reacts with DQwl, DQ blank, and some DQw3 specificities (the so-called DR4/DQw3.2 and DRw9/DQw3.3; see Figure 1) [2,4,9, 11,15]. There is evidence that the TA10/2B3 specificities reside on the DOff chains [2,4,15]. In order to identify sequences perfectly correlated with TA10/2B3 specificities, we made a full-scale comparison of the available HLA-DQ,8 sequences [ 16-36]. These comparisons led to the design of a set of oligonucleotide probes hybridizing to codon 45 and adjacent nucleotides on the Doff gene that appears to be TA10/2B3-specific. These findings were confirmed by panel and family segregation studies.

From the Department of lmmunohaematology and Blood Bank, University Hospital, Leiden, The Netherlands. Address reprint requests to." Mr. M. J. H. Kenter, Leiden University Hospital, Department of lmmunohaematology and Blood Bank, Building 1, E3-Q, P.O. Box 9600, 2300 RC Leiden. The Netherlands. Received June 30, 1988: revised September 7, 1988.

Human Immunology 24, 65-73 (1989) © American Society for Histocompatibility and Immunogenetics, 1989

65 0198=8859/89/$3.50

10

20

30

40

~

50

60

70

80

F...G

F...G F...G

........

........ ........ ............ ............

~

............

DR2/RZH/DQuI

DRw6/DuS/DQul DRu6/DuIS/DQul

...........

GL . . . . . . . . . . .

GL . . . . . . . . . . .

~

............ ............ G ............ G ............

.........

G ............

~

......

~

V......

U......

U ......

IV ...... IV ......

.............

G...H ......

G...H ......

G...H

G .................. G .................. L.S.S ..... L.S.S .....

..................

. ..L...H

L..G

~

L ..................

L

Q.R Q.R.V

G .......

~ ..........

......

......

OI ................

G .............

G ........

GR..5U.R

GR..5U.R

..........................

..........

Q.R

..............

..............

Q.R

G .......

~

90

EUGY.

EVfF.

EVRF.

EVRY.

GI ....

GI

GI

GI ....

EUFIY. G I

DI..ED..SU ................... D I . . E D . . SV . . . . . . . . . . . . . . . . . . . DI..ED..5U ................... DI...K..RV.R ................. DI...K..RU.R ................. GR..SV.R ...... EVRY. G I . . . .

............

..............

.Q.R

.Q.R.S

~

R .....................................

~ ...RL ......... . . . RL . . . . . . . . . ...RL ......... L..L.R ........ L..L.R ........ .Q.R.U ............

.....

G .......

G .......

G. . . . .

G. . . . .

G .....

G ..... G ..... GEF.. GEF...

G .....

G .....

~

FIGURE 1 Alignment of HLA-DQ~ first domain amino acid sequences: DR4/DQw3.1; DR5/DQw3.1, DRw9/DQw3.3, DRw8/DQ blank, and DRw6/DwlS/DQwl [ 11]; DR4/ DQw3.2 [25]; DRw8/DQw3.1 [20]; DR4/DQ blank. [18]; DR3/DQw blank [22]; DR3/ DQw2 [16]; DRT/DQw2 [23]; DR1/DQwl [34]; DR2/AZH/DQwl, DR2/Dw2/DQwl, and DR2/Dw12/DQwl [26]; DRw6/Dw9/DQwl [32]; DRw6/Dw19/DQwl [3,21]. The TA10-specific amino acid residue at position 45 is marked by an arrowhead. The IIB3/ TA10 reactivities are indicated; ÷ = positive, - = negative. The IIB3 low affinity specificities as previously described [2,9,13] are marked by ±.

........ F...G ............ L ................. DR2/Dw2/DQwl - .P ..... L ........................... DU ...... DR2/DuI2/DQwl G ............ L...H .............. D R w 6 / D u I Y / D Q w l ............

............

............

DRI/D~I

............

~

R~PEDF~YgFKRMCYFTNGTE~RYUTRYIYNREEY~RFD~UE~YRR~TPLG~P~REYWNS~KE~LERTRRELDT~CRHNYgLELRTTL~RR

+l

DR5/DQw3.! DRwB/DQw3.1 DRu9/DQw3.3 DR4/DQu3.2 DR4/Dg-bIank DR=B/DQ-blan~ DR3/DO-blank DR3/DQu2 DRT/Dgu2

DR4/DQw3.1

÷

-

-

-

÷

÷

÷

÷

÷

-

÷

-

÷

-

÷

-

÷

÷

÷

antibody reactivity TRIO lIB3

G~ G~

TA10 and 2B3 Oligonucleotide Typing TABLE 1

67

Sequences of oligonucleotide primers and probes a

Specificity

Sequence

Ref

TA10 2B3 DQw2 5' DQ/3 SalI 3' DQfl XbaI

5'-GACAGCGACGTGGAGGTGTA 5'-GACAGCGACGTGGGGGTGTA 5'-GGGGAGTTCCGGGCGGTGAC 5'-CCGGTCGACTCCCCGCAGAGGATTTCGTG 5'-TGCTCTAGAGGGCGACGACGCTCA~CTC

29 25 16,23 16,25,29 16,25,29

a The sequences were derived from the indicated references. The oligonucleotide primers anneal to the HLA-DQfl exon II and parts of its flanking intron sequences. The TA10 and 2B3 olignucleotide probes hybridize to the DQB region encoding amino acids 41-47 and differ in one nucleotide as indicated. The HLA-DQw2 oligonucleotide probe anneals to the region coding for amino acids 45-51.

MATERIALS A N D M E T H O D S

Cell lines. The panel contains both local and Tenth Workshop homozygous cell lines, which were extensively studied by others and by us [1,2,10,11 and references therein, 13,37].

Serologic typings. HLA typings were performed with standard tests as previously described [9,37].

Oligonucleotides. Oligonucleotides (Table 1) were synthesized by the /3-cyanoethyl amidite method (New Brunswick cyclone). The oligonucleotide probes were end-labeled using [3,-32p]-ATP (5000 Ci/mmol Amersham, U.K.) and T4polynucleotide kinase according to Bos et al. [38]. Oligonucleotide primers were designed, including an artificial restriction site at the 5' end for cloning purposes.

Amplification of genomic DNA. DNA was isolated from 3~lymphoblastoid cell lines (B-LCLs) as described previously [10]. Amplification of specific sequences was performed by the polymerase chain reaction (PCR) technique with Taqpolymerase [39] according to a method slightly modified from Saiki et al. [40]. Reactions of 100 /~l typically contained 1 /zg of genomic DNA in 16.6 mM ammonium sulfate, 67 mM Tris-HCl (pH 8.8), 6.7 mM magnesium chloride, 10 mM /3-mercaptoethanol, 6.7 ktM EDTA, 170 /zg bovine serum albumin per milliliter, 0.5 mM each dNTP, 10% (v/v) DMSO, and 40 pmol of each primer, overlaid with 1 vol paraffin oil. After heating for 7 min at 96°C and cooling down to 37 ° for 5 min, 5U ofTaq-DNA polymerase (Perkin, Elmer, and Cetus) were added. Next the samples were subjected to 40 cycles of amplification by heating for 1 min at 96°C, cooling for 10 s at room temperature, followed by an incubation at 56°C for 2 min. During the amplification no additional Taq-DNA polymerase was added. After the amplification procedure the samples were spun down, and paraffin oil was removed carefully.

Hybridization of amplified DNA with oligonucleotide probes. Typically, 5/.d of the amplified sample was denatured in 100 /zl 0.4 M NaOH, 25 mM EDTA and loaded onto prewetted Genescreen Plus (NEN) membranes using a dot blotter (Minifold, Schleicher, and Schuell). Next the wells were washed with 20x saline sodium phosphate EDTA (SSPE) (1 x SSPE is 150 mM NaC1, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4) and the filters were UV-irradiated for 4 min to bind the

68

M.J.H. Kenter et al.

1

12

A

C

FIGURE 2 A representative example of DQ,8 DNA oligonucleotide typing for the TA10 (A), 2B3 (B), and DQw2 specifcity (C) on DQ,8 PCR-amplified DNA samples of the following B-LCLs: #1 CJO, # 2 0 0 S , #3 MVL, #4 1WB, #5 VYF, #6 SAS, #7 AZH, #8 QBL, #9 AVL, #10 CAA, #11 HAR, #12 WEN. See Table 3 for the HLA typing of these B-LCLs and Table 1 for the oligonucleotide sequences.

DNA. The filters were prehybridized for 1 hr at 37°C in 5 x SSPE, 5 × Denhardt solution, 0.5% (w/v) sodium dodecyl sulfate 100/.tg/ml herring sperm, and hybridized overnight with 32p-end-labeled oligonucleotide probes (at 2 × 105 cpm/ ml). Blots were washed at 61°C in a tetramethylammonium chloride solution as described by Wood et al. [41] and exposed for 1 hr at -80°C to Kodak XAR-5 films using an intensifying screen. RESULTS A N D DISCUSSION A comparison of the DQw3.1/3 chains (TA10-positive) with the DR4/DQw3.2 (2B3-positive) /~ chain sequence shows four amino acid differences between these two alleles, at positions 13, 26, 45, and 57 (Figure 1). A more extensive inspection of the available published HLA-DQ3 protein sequences revealed that only the amino acids at position 45 and its immediate surroundings are perfectly correlating with either TA10, 2B3, or HLA-DQw2 reactivity (Figure 1). To test whether this particular region indeed contributes to the TA10/2B3 determinants, we hybridized PCR-amplified DQ/3 DNA with three specific oligonucleotide probes (Table 1; Figure 2). A panel of B-LCLs was screened to compare the known antibody reactivity with the oligonucleotide probe hybridizations (Figure 2; Table 2). The probe hybridizations correspond precisely with the reactivity of the TA 10/IIB3 antibodies. One family study was performed (Table 3). Since the mother is homozygous HLA-DR3/DQw2 (TA 10/2B3-negative), all

TA10 and 2B3 Oligonucleotide Typing TABLE 2

69

A homozygous typing cell panel study of the TA10 and 2B3 specificities by serology and HLA-DQB DNA oligonucleotide typinga DQfi Antibody

DNA

reactivity

oligotyping

Cell line

HLA-DR

-Dw

-DQw

TA10

IIB3

TA10

2B3

CJO

1

1

1

-

+-

-

+

OOS

1

1

1

-

-+

-

+

MVL

1

1

1

-

-+

-

+

IWB

2

2

1

-

+

-

+

VYF

2

2

1

-

+

-

+

SAS

2

12

1

-

+

-

+

AZH

2

AZH

1

-

_+

-

+

18

1

-

+

-

+

HHK

w13

KRA

w13

19

1

-

+

-

+

BRU

w14

9

1

-

-+

-

--

QBL

3

3

2

-

AVL

3

3

2

-

-

-

CAA

3

3

2

-

-

HAR

3

3

2

.

.

.

.

EKR

7

7

2

-

-

-

BSM

4

4

3

-

+

-

MWM

4

4

3

+

-

+

LRM

+

4

4

3

+

4

4

3

+

JB10

4

10

3

PEE

4

13

3

LS40

4

14

3

-

MT

4

14

3

-

YT2

4

15

Blank

-

+-

-

+

YOT3

4

15

Blank

-

-+

-

+ -

WEN

b

+

+

+

-

+

+

+

-

+

-

+

-

+

-

+

+

-

+

ATH

w 11

5

3

+

-

+

THR

wl 1

5

3

+

-

+

-

JVM

w 11

LDVII

3

+

-

+

-

HLF

w12

DB6

3

+

-

+

SWEIG

wl 1

5

3

+

-

+

-

CRI

w14

16

3

+

+

-

LIA

w14

16

3

+

-

+

-

LUY

w8

8

3

+

-

+

-

MMR

w8

8

-

-+

-

+

DKB

w9

DB5

-

+

-

+

Blank 3

" Reactivities of the IIB3/TA10 MoAbs are indicated: + = positive, s p e c i f i c i t i e s as p r e v i o u s l y d e s c r i b e d [ 2 , 9 , 1 3 ] a r e m a r k e d b y + . b The cell line WEN

is h e t e r o z y g o u s

for the TA10

= n e g a t i v e r e a c t i o n . T h e l I B 3 Low affinity

a n d I I B 3 s p e c i f i c i t i e s as p r e v i o u s l y d e s c r i b e d [ 1 , 1 0 , 3 7 ] .

the children are HLA-DQw2-positive. The father is TA10/2/33-positive; therefore the children are either TA10 (children 1, 2, and 3)- or 2B3-positive (children 4 and 5). Just as in the panel study, the hybridization patterns of our selected probes on PCR-amplified genomic D N A from this family correlated exactly with TA10/2B3 antibody typing data.

70

M.J.H. Kenter et al. TABLE 3

Family segregation study of the TA10 and 2B3 specificities by serology and DQg DNA oligonucleotide typing Antibody

DQB

reactivity

DNA oligotyping

HaploDR

DQw

type a

TA10

lIB3

TA10

2B3

DQw2

+

±

-

-

+

-

+

Father

414

3/3

a/b

+

+

Mother

3/3

2/2

c/d

-

-

Child 1

3/4

2/3

a/c

+

-

t

Child 2

3/4

2/3

aid

+

-

+

Child 3

3/4

2/3

a/d

+

-

+

-

Child 4

3/4

2/3

b/c

-

+

-

+

+

Child 5

3/4

2/3

b/d

-

+

-

~

+

+ +

The HLA haplotypes are: a = A 2 , B w 5 2 , C w - , DR4, DQw3, TA10; b = A3, B37, Cw6, DR4, DQw3, 2B 3; c A2, Bw35, Cw4, DR3, DQw2; and d = A I , BS, Cw7, DR3, DQw2.

Conflicting data exist on the DNA sequence encoding amino acid 45 of the DO.fl3.1 chain of the homozygous B-LCL Sweig [20,31]. Our results agree with the data by Holbeck and Nepom [20], namely, that codon 45 is GAG (Glu) instead of reported GGG (Gly). This is concluded since PCR-amplified DQ/~ DNA from the B-LCL Sweig hybridizes selectively with our TA10 (GAG) and not with our 2B3 oligo (GGG) (Table 2). Moreover, all other published DQw3.1 sequences show a glutamic acid instead of a glycine at position 45 of the DQ3 chain [3,20,29,33]. Recently, oligonucleotide probes hybridizing to a region near position 26 of the DQ3 chain sequence were reported that were claimed to type for the DQw3.1 specificity on Southern blots [33,42]. On comparing the DQfl sequences available ([16-36] and Figure 1), it can be concluded that there is no TA10-specific sequence located near that region. Consequently, these probes do not allow unambiguous TA10 oligonucleotide typing on PCR-amplified DNA. To our knowledge, no 2B3-specific oligonucleotide probes have been described before. Our findings indicate that the negatively charged glutamic acid at position 45 of the DQ,8 chains is essential for the TA10 specificity and that all 2B3-positive DQg chains bear the sequence Gly-Val-Tyr (codons 45-47). TA10 and 2B3 oligonucleotide probes make it possible to type unambiguously for the TA 10 and 2B3 specificities at the DNA level and can be used in a routine way. The affinity problems associated with the use of the MoAb IIB3 for the HLA-DR1/DQwl, DR2/AZH/DQwl, DRw6/Dw9/DQwl, and the DQ blank specificities in serology [2,9,13] can thus be avoided (Table 2). Therefore, the 2B3 oligotyping is a valuable alternative for serologic 2B3 typing. The TA10 and 2B3 oligonucleotides may be relevant for disease association studies in which the TA 10 and/or 2B3 markers are thought to be involved, such as studies of insulin-dependent diabetes mellitus and rheumatoid arthritis [37,43-46].

ACKNOWLEDGMENTS

We thank Dr. H. Erlich, Dr. H. Bos, and Mr. S. Scharf for helping us with the PCR technique, the laboratories that kindly provided HLA homozygous typing cells, Mr. T.

TA10 and 2B3 Oligonucleotide Typing

71

Rinke de Wit, Drs. R. Bontrop, P. van den Elsen, and J. D'Amaro for critical reading of this manuscript and helpful discussions, and Mr. A. M. I. H. Naipal for excellent technical assistance. This work was partially supported by the Dutch League Against Rheumatism and the Institute for Radiopathology and Radioprotection.

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M . J . H . Kenter et al. 15. Radka SF, Scott RG, Stewart SJ: Molecular complexity of HLA-DQw3: The TA 10 determinant is located on a subset of DQw3 beta chains. Hum Immunol 18:287, 1987. 16. Boss JM, Strominger JL: Cloning and sequence analysis of the human major histocompatibility complex gene DC-3-beta. Proc Natl Acad Sci USA 81:5199, 1984. 17. G6tz H, Kratzin H, Thinnes FP, Yang C, Kruse T, Pauly E, K61bel S, Egert G, Wernet P, Hilschmann N: Primary structure of human class II histocompatibility antigens. Hoppe Seyler's Z Physiol Chem 364:749, 1983. 18. Gregersen PK, Shen M, Song Q-L, Merryman P, Degar S, Seki T, Maccari J, Goldberg D, Murphy H, Schwenzer J, Wang CY, Winchester RJ, Nepom GT, Silver J: Molecular diversity of HLA-DR4 haplotypes. Proc Natl Acad Sci USA 83:2642, 1986. 19. Hilden JM, Curtsinger JM, Cairns JS, Bach FH: DQ beta sequences in HLA-DR4 haplotypes. Hum lmmunol 18:261, 1987 20. Holbeck SL, Nepom GT: Molecular analysis of DQ-beta3.1 genes. Hum Immunol 21:183, 1988. 21. Horn GT, Bugawan TL, Long CM, Manos MM, Erlich HA: Sequence analysis of HLA class II genes from insulin-dependent diabetic individuals. Hum Immunol 2l:249, 1988. 22. Hurley CK, Gregersen P, Steiner N, Bell JI, Hartzman R, Nepom G, Silver J, Johnson AH: Polymorphism of the HLA-D region in American blacks: A DR3 haplotype generated by recombination. J Immunol 140:885, 1988. 23. Karr RW, Gregersen PK, Obata F, Goldberg D, MaccariJ, Alber C, Silver J: Analysis of DR-beta and DQ-beta chain cDNA clones from a DR7 haplotype. J Immunol 137:2886, 1986. 24. Larhammer D, Schenning L, Gustafsson K, Wiman K, Claesson L, Rask L, Peterson PA: Complete amino acid sequence of an HLA-DR antigen-like beta chain as predicted from the nucleotide sequences: Similarities with immunoglobulins and HLAA, -B, and -C antigens. Proc Natl Acad Sci USA 79:3687, 1982. 25. Larhammer D, Hyldig-Nielsen JJ, Servenius B, Andersson G, Rask L, Peterson PA: Exon-intron organisation and complete nucleotide sequence of a human major histocompatibility antigen DC-beta gene. Proc Natl Acad Sci USA 80:7313, 1983. 26. Lee BSM, Bell JI, Rust NA, McDevitt HO: Structural and functional variability among DQ-beta alleles of DR2 subtypes. Immunogenetics 26:85, 1987. 27. Liu C-P, Bach FH, Wu S: Molecular studies of a rare DR2/LD-5a/DQw3 HLA class II haplotype: Multiple genetic mechanisms in the generation of polymorphic class II genes. J Immunol 140:3631, 1988. 28. Lock CB, So AKL, Welsh KI, Parkes JD, TrowsdaleJ: MHC class II sequences of an HLA-DR2 narcoleptic. Immunogenetics 27:449, 1988. 29. Michelsen B, Lernmark A: Molecular cloning of a polymorphic DNA endonuclease fragment associates insulin-dependent diabetes mellitus with HLA-DQ. J Clin Invest 79:1144, 1987. 30. Schenning L, Larhammer D, Bill P, Wiman K, Jonsson A-K, Rask L, Peterson PA: Both alpha and beta chains of HLA-DC class II histocompatibility antigens display extensive polymorphism in their aminoterminal domains. EMBO J 3:447, 1984. 3 I. Schiffenbauer J, Didier DK, Klearman M, Rice K, Shuman S, Tieber VL, Kittlesen DJ, Schwartz BD: Complete sequence of the HLA DQ-alpha and DQ-beta cDNA from a DR5/DQw3 cell line. J Immunol 139:228, 1987.

TA10 and 2B30ligonucleotide Typing

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