Eur. J. Immunol. 1990. 20: 1603-1606
Analysis of HLA-DR p l chain in HAM
1603
Short paper Koichiro UsukuO, Masatoyo NishizawaO, Kazumasa MatsukP, Katsushi TokunagaO, Keikichi TakahashiO, Nobutaka Eiraku., Masahito S u e h a d , Takeo JujiO, Mitsuhiro Osame. and Takeshi TabiraO Division of Demyelinating Disease and Agingo, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Blood Transfusion Servicen, Tokyo University Hospital, Tokyo and Third Department of Internal Medicine., Kagoshima University, Kagoshima
Association of a particular amino acid sequence of the HLA-DR p l chain with HTLV-I-associated myelopathy" Analyses of HLA-DRB1 gene using polymerase chain reaction and sequencespecific oligonucleotide probes reveal that the amino acid sequence Glu-Gln-Arg-Arg-Ala-Ala-Val at positions 69-75 of the third hypervariable region (HVR) of HLA-DR p l chain is significantly associated with HTLVI-associated myelopathy (HAM). Since the frequency of this sequence in HTLV-I carriers is almost the same as that in controls from Kagoshima (an endemic area of HTLV-I), this sequence may be related to susceptibility to HAM rather than to susceptibility to HTLV-I infection. Since this third HVR functions as putative antigen-binding sites and T cell recognition sites, the amino acid sequence of positions 68-73 was analyzed in detail. The analysis reveals that Gln'O and Arg'l are relevant to the occurrence of HAM.
1 Introduction Human Tcell lymphotropic virus type I (HTLV-I) causes at least two diseases, HTLV-I-associated myelopathy (HAM) [ 1]/HTLV-I-associated tropical spastic paraparesis (HTLVI/TSP) [2] and adult Tcell leukemia (ATL) [3]. The viruses isolated from patients with HAM and ATL are identical by the restriction enzyme analysis [4, 51 and by the nucleotide sequence analysis [6]. However, it is not known how HTLV-I induces two different diseases. Our previous study has demonstrated that HAM and ATL are associated with different HLA haplotypes; the frequency of HLA-DR1 and -DR4 is higher in HAM and the frequency of HLA-A26 is higher in ATL [7]. A strong association between acute type of ATL and HLA-Bw62Cw3-A26 haplotype is reported [8]. Furthermore, in HAM patients, a higher immune response to HTLV-I is demonstrated than in ATL patients [7]. These data suggest that HAM and acute type of ATL occur in two different populations, and that HAM is related to HLA-DR and ATL to HLA class I. Recent advances in technology of HLA typing make it possible to examine disease susceptibility through determi-
nation of specific amino acid (aa) sequence(s) [9-121, which may be shared among several alleles. In this report, we have focused on the p-chain sequence of the DR molecule to investigate which part of this chain is important for the occurrence of HAM. For this purpose, a specific segment of the HLA-DRB1 gene in human genomic DNA was amplified using polymerase chain reaction (PCR) and the sequence variation was analyzed with sequence-specific oligonucleotide (SSO) probes. We show a definite association of a particular D R p l chain sequence with HAM,which contributes to HAM susceptibility.
2 Materials and methods 2.1 Patients and control subjects For this study 32 patients with HAM, 8 patients with ATL, 46 asymptomatic HTLV-I carriers, 40 unrelated controls residing in Kagoshima, a prefecture in the southwestern Japan where HAM and ATL are prevalent [13], and 36 unrelated controls residing inTokyo, a non-endemic area of HTLV-I infection, were examined. All Tokyo controls were previously HLA typed. 2.2 Oligonucleotide primers and SSO probes
[I 81131
*
This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture of Japan.
Correspondence: Masatoyo Nishizawa, Division of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, Japan Abbreviations: HTLV-I: Human T cell lymphotropic virus type I HAM: HTLV-I-associated myelopathy TSP: Tropical spastic paraparesis ATL: Adult Tcell leukemia PCR: Polymerase chain reaction SSO: Sequence-specific oligonucleotide aa: Amino acid(s) HVR: Hypervariable region 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
The DRBl oligonucleotide primers for PCR were GLPDRBl (5'-ITCTTCAATGGGACGGAGCG-3') and GAMPDRBl (5'-GCCGCTGCACTGTGAAGCTCTC3') which anneal t o the region of exon 2 of the DRB gene encoding the aa 17 to 23 and aa 87-94, respectively (Fig. 1). SSO probes have been designed to investigate specific aa sequences on HLA-DR p l chain in this study. Table 1 summarizes the nucleotide sequence of each probe, the aa position and sequence encoded by each probe and HLA alleles reported to include each aa sequence. To ensure the specificityof each probe, serologically typed standard DNA samples were amplified, slot blotted and hybridized simultaneously with test samples, and each spot on the blot was determined as positive or negative. 0014-2980/90/0707-1603$03.50+ .25/0
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Eur. J. Immunol. 1990. 20: 1603-1606
K. Usuku, M. Nishizawa, K. Matsuki et al.
Table 1. Synthesized oligonucleotide probes
Probe 1 2 3 4 5C) 6
7 8
Oligonucleotide Sequencea)
aa sequence
THb)
T AC T T C T A T CA C CAAGAGGA CGGCCTAGCGCCGAGTAC GCAGAGGCGGGCCGCGGT CTGGAGCAGAGGCGGGCC
3UyFYHQEE36 "RPSAEY 6YEQRRAAV5 68LEQRRA73
48
. . . .
. . . .
. . . .
. . . .
.AG.C . . .AG.CGA .AG.C . . . . .G . . .
. . .C . . . . . . . . . .
("C)
68LEDRRA73 68LEDERA73 68LEDRRA73 6BLERRRA73
. . . . . . . . . . . . . .
63 70
66 64 63
64 73
Reported HLA allele DR4 DR4Dwl5,DRwS DR1,DR4Dwl4/15,DRwl4Dwl6 DR1,DR4Dw13/14/15/KT2, DRw14Dw16 DRw15DwU12 DR4DwlO,DRwl3 DRSDw5,DRwS DRw14Dw9,DRw9,DRw10
a) Each nucleotide sequence is written in order of 5'- to 3'-end. b) TH, temperature ("C) for hybridization. c) Periods indicate identity of the nucleotide to that of probe 4.
10
20
W1 CW4M4 W4Dw15 DRwl4Dwl6........EY CW4Dwl3 DR4DKT2
........ E. V.H....... ........E. V.H....... STS....... ........ E. V.H....... ........E. V.H....... ........Q. D.Y.......
DliWl6Dw2 DFMl6Dwl2........Q.
D.Y.......
R
40
..... F.D.Y .....F.D.Y
F.H...Y... F.H...Y... F...Y FH.... N... .....F. D.Y F.H...Y... F.D.Y F.H.......
..... .....
.....F.H.D ......DL.. ..... F.H.G ......N...
SIS....... Y....
SIS....... D......... V.........
.....F.D.Y FH.... Y... ..... Y.H.G ......N... ......... R VH.... YA.Y
m
........EY
DRw8
........EY
50
w IyNgEEsvRFV-
SIX..
........ E. V.H....... ........EY STS....... ...
E
.....FF..DD..YY ..... .....FF..DD..YY .....
lX4DwlO IXwl3
DRw14Dw9 ...EY DRw9 Q....K. CWwlO ........EE.
30 l
F.H...Y... FH.... N... F.....Y... F.....Y...
60 70 80 !TECGRPDAEY WSXFDLLEQ RR?AVtYlYG?
90 UBR
............................................. V.... .... ................ S... .................................. ...................................................... ................................. EE............ ..... ................................. .....VV........ .... .......................... FF....DD ........................ .......................... .................... .......................... I..D E......... .....V.... .... ......F... ................I..D E.,....... .....V.... .... ......F... .......E.. ......F..D ........................ ................ S... ......F..D ...L...... ...... ................A.DD .........R ...E...... .....V.... .... ................ V..S ......F..R ...E...V.. ...... ............................. R ........................
Figure 1. aa sequences (in one-letter code) of the first domain of the HLA-DR p l chain.The numbers above sequence refer to positions of the aa. Periods indicate identity with the DR1 sequence. Blanks indicate that no sequence information is available. Arrows indicate positionsof PCR primers. References of the sequences are as follows: DR4Dw13 from Cairns et al. [14];DR4Dw15 from Gregersen et al. [15]; DRwlOfrom Merryman et al. [16]; DRwl4Dw9 from Gorski and Mach [17]; DRw14Dw16from Gorski [18]; the others fromTodd et al. [9].
2.3 In vitro DNA amplification
2.4 Dot blotting and detection of specific sequence
Genomic DNA was extracted from the white blood cells of patients and controls as described previously [19]. PCR was carried out in 100 pl of the reaction mixture containing 1-5 pg genomic DNA, 10 mM Tris-HC1, pH 8.3, 50 mM KCI, 1.5 mM MgC12, 200 p~ of dATP, dlTP, dGTP and dCTP, and 1 pg of each primer. The sample was heated at 95 "C for 10 min, cooled to 50°C and 2.5 units of Taq DNA polymerase was (Perkin Elmer Cetus Corp., Norwalk, CT), then subjected to a thermal cycle with 72 "C for 2 min, 95 "C for 1min and 50 "C for 2 min. This procedure was repeated 30 times, then followed by an additional incubation at 72 "C for 5 min to complete the extention. After completion of the amplification, 6 pl of the sample was electrophoresed to confirm the amplification.
The amplified DNA sample was mixed with 530 pl of 0.5 M NaOH and 25 mM EDTA, then 200 pl of this mixture was blotted onto nylon filter (Zeta-Probe Blotting Membranes, Bio-Rad, Richmond, CA). The hybridization using each probe was carried out at the temperature summarized in Table 1. Each filter was prehybridized in a bag containing 5 X SSPE (720 mM NaCl, 40 mM NaH2P04,4 mM EDTA, pH 7.4), 5 x Denhardt's solution (0.1%BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone) and 5% SDS for 1 h at the hybridization temperature. The SSO probe (10 pmol) was 3' end-labeled with [a-32P]dideoxyATP(= 111MBq), and purified through a Sephadex column (NAPS; Pharmacia, Uppsala, Sweden). After prehybridization the 32P-labeled probe was added to the prehybridization buffer, and the
Analysis of HLA-DR fJ1 chain in HAM
Eur. J. Immunol. 1990. 20: 1603-1606
filter was hybridized for additional 1 h. The filter was then washed once in 2 x SSPE/2.5% SDS for 10 min, and once in 2 X SSPE/1.0% SDS for 10 min at the hybridization temperature, and then exposed with an intensifying screen at - 8 5 O C f o r 6 h t o 2 4 h .
Probe 2
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Probe 3
2.5 Statistical analysis Statistical analysis was done using the x2 test or the Fisher’s exact test.
3 Results Since HLA-DR1 and DR4 were frequently seen in patients with HAM [7], we first analyzed HLA-DR1 and DR4 using SSO Probes 1 to 3 (Table 2). Representative blots are shown in Fig. 2. The analysis shows that frequency identified positive by probe 3 (HLA-DR1, DR4Dw14/15, DRw14Dw16) in HAM patients is significantly higher than in ATL patients (p < 0.04, relative risk 6.60), HTLV-I carriers (p < 0.05, relative risk 2.86), controls in Kagoshima (p < 0.02, relative risk 3.30) or controls in Tokyo (p < 0.00005, relative risk 10.0). It is also noted that controls in Kagoshima have a higher frequency than those in Tokyo (p < 0.05, relative risk 3.30). Since allelic specificity of HLA-DR haplotype in the third hypervariable region (HVR) of HLA-DR p l chain can be classified into five groups in case of the Japanese, five SSO probes have been designed to further examine the aa difference in detail among aa residues 68-73 (HLA-DRwl6Dw21, DR3, DR4Dw4 and DR7 are known to be extremely rare among the Japanese [20], and have, therefore, been omitted in this study). Table 3 summarizes that only probe 4 (HLA-DR1, DR4Dw13/14/15/KT2, DRw14Dw16) showes significantly higher frequency in HAM patients than in ATL patients (p < 0.05, relative risk 7.67), HTLV-I carriers (p < 0.01, relative risk 3.67) or
Figure2. Representative slot blot of 20 patients with HAM. A specific segment of the HLA-DRB1 gene was amplified by the PCR method and hybridizid with probe 2 and probe 3.The number written on the left of each spot is an identity number of each HAM patient.
Kagoshima controls (p < 0.01, relative risk 3.83). Other probes do not show significant positive or negative association with HAM.
4 Discussion Recent studies of specific HLA class I1 sequences in patients with some autoimmune diseases and controls have provided more informative markers for genetic susceptibility, and suggested new strategies for clarifying pathomechanisms of the diseases [9, 101. A T cell recognizes a foreign antigen through interactions among the TcR, processed antigen fragments and the polymorphic residues of
Table 2. Dot blot analysis of HAM patients, ATL patients, HTLV-I carriers and controls
HAM ATL HTLV-I carrier Kagoshima control Tokyo control
n
Probe 1
Probe 2
Probe 3=)
32 8
13 (40.6)b) 3 (37.5) 20 (43.4) 15 (37.5) 11 (30.6)
14 (43.8) 2 (25.0) 23 (50.0) 20 (50.0) 12 (33.3)
22 (68.8) 2 (25.0) 20 (43.5) 16 (40.0) 6 (16.7)
46 40 36
a) Probe 3: HAM vs. ATL, relative risk (RR) = 6.60, x2 = 3.444, p < 0.04; HAM V S . HTLV-I camer, RR = 2.86, x2 = 4.856, p < 0.05: HAM vs. Kagoshima control, RR = 3.30. x2 = 5.896, p < 0.02; HAM vs. Tokyo control, RR = 10.0, x2 = 18.97, p < O.ooOo5: Kagoshimacontrol vs.Tokyocontro1, RR = 3.30. x2 = 5.015, p < 0.05. b) Percentages are shown in parentheses.
Table 3. Dot blot analysis of aa sequence 68-73 of HLA-DR p l chain in patients with HAM, patients with ATL, HTLV-I carriers and controls a) Probe 4: HAM vs. ATL, relative risk (RR) = 7.67, x* = n Probe4a) Probe5 Probe6 Probe 7 Probe8 4.167, p < 0.05; HAM vs. HTLV-I carrier, RR = 3.67, HAM 32 23 (71.9)b) 7 (21.9) 4 (12.5) 7 (21.9) 12 (37.5) = 7.210, p < 0.01: HAM vs. ATL 8 2 (25.0) 3 (37.5) l(12.5) 3 (37.5) 3 (37.5) Kagoshima control, RR = 3.83, 8 (19.0) HTLV-I carrier 42 17 (40.5) 17 (40.5) 3 (7.1) x2 = 7.276, p < 0.01. 22 (52.4) 14 (35.0) 2 (5.0) Kagoshima control 40 16 (40.0) 5 (12.5) 18 (45.0) b) Percentages are shown in parentheses.
x2
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K. Usuku, M. Nishizawa, K. Matsuki et al.
HLA class I1 molecules [21]. HAM shows several abnormal immune reactions probably mediated by activated T cells [22]. It is our aim to elucidate genetic susceptibility to HAM, and to clarify HLA class I1 sequences which may play important roles in the occurrence of HAM, through determination of specific aa sequences rather than conventional serological allelic typing. The present study reveals that the aa sequence EQRRAAV at positions 69-75 of the HLA-DR p l chain identified by probe 3 is significantlyassociated with HAM. Although the frequency of this sequence in HAM is the highest, it is almost the same between HTLV-I carriers and Kagoshima controls (Table 2). This may indicate that the aa sequence EQRRAAV is related to susceptibility to HAM rather than to susceptibility to HTLV-I infection. The difference in frequencies of this sequence observed in HAM and ATL patients is attributable to the difference in their respective genetic background [7]. The difference of frequency of this sequence in Kagoshima controls and Tokyo controls may be related to the geographical preponderance of HAM in Kagoshima [13]. According to the model of class I1 molecule proposed by Brown et al. (231, aa residues at positions 70 and 71 are within the putative antigen-binding site of HLA-DR b l chain a-helix. Highly polymorphic regions of the a-helix include those functioning also as T cell recognition sites [lo, 241. In particular, the third HVR of the HLA-DR fi1 chain, aa positions 65-75, includes not only an antigenbinding site [23] but also a target of alloreactiveTcel1 clones [25].We then examined the aa sequences of positions 68-73 in detail, because difference in the aa sequence in this region is determined by positions 70 and 71. As shown in Table 3, frequency of the aa sequence LEQRRA is significantly higher in HAM patients than in ATL patients, HTLV-I carriers or Kagoshima controls. This may indicate that the aa residues Gln7" and Arg71 are important in the occurrence of HAM. Furthermore, we have not found any particular aa residues or sequences in this region that are negatively associated with HAM, contributing to resistance to HAM. The results of the aa sequence analysis in rheumatoid arthritis (RA), a typical autoimmune disease [lo-121, clearly show that the aa sequence in the third HVR of HLA-DR1 and of some of HLA-DR4 haplotypes has positive association with RA. It is of interest to know that HAM and RA have association with the same HLA class I1 aa sequence. Although HTLV-I infection is the cause of HAM, the pathomechanism is still obscure. Nevertheless, it is important to know that there is a relationship between the particular aa sequence of HLA-DR p1 chain and HAM, and this has been accomplished by investigating aa sequences among different HLA-DR alleles. Further analyses are essential to reveal antigen(s) and T cells contributing to the occurrence of HAM.
Eur. J. Immunol. 1990. 20: 1603-1606 Received, November 16, 1989; in revised form March 12, 1990.
5 References 1 Osame, M., Matsumoto, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H.,Tara, M. and Igata, A., Ann. Neurol. 1987. 21: 117. 2 Gessain, A., Barin, E , Vernant, J. C., Gout, O., Maurs, L., Calender, A. and deThe, G . , Lancet 1985. ii: 407. 3 Yoshida, M., Miyoshi, I. and Hinuma,Y., Proc. Natl. Acad. Sci. USA 1982. 79: 2031. 4 Yoshida, M., Osame, M., Usuku, K.. Matsumoto, M. and Igata, A., Lancet 1987. i: 1085. 5 Imamura, J.,Tsujimoto, A., Ohta,Y., Hirose, S., Shimotohno, K., Miwa, M. and Miyoshi, I., Int. J. Cancer. 1988. 42: 221. 6 Tsujimoto, A., Terauchi, T., Imamura, J., Shimotohno, K., Miyoshi, 1. and Miwa. M., Mol. Biol. Med. 1988. 5: 29. 7 Usuku, K., Sonoda, S., Osame, M.,Yashiki, S.,Takahashi, K., Matsumoto, M., Sawada,T.,Tsuji, K.,Tara, M. and Igata, A., Ann. Neurol. 1988. 23: 143. 8 Uno, H., Kawano, K., Matsuoka, H. and Tsuda, K., Clin. Exp. Immunol. 1988. 71: 211. 9 Todd, J. A., Bell, J. I. and McDevitt, H. O., Nature 1987. 329: 599. 10 Todd, J. A., Acha-Orbea, H., Bell, J. I., Chao, N., Fronek, Z., Jacob, C. O., McDermott, M., Sinha, A. A.,Timmerman, L., Steinman, L. and McDevitt, H. O., Science 1988. 240: 1003. 11 Watanabe,Y.,Tokunaga, K., Matsuki, K.,Takeuchi, F., Matsuta, K., Maeda, H., Omoto, K. and Juji,T., J. Exp. Med. 1989. 169: 2263. 12 Wordsworth, B. P., Lanchbury, J. S. S., Sakkas, L. I.,Welsh, K. I., Panayi, G. S. and Bell, J. I., Proc. Natl. Acad. Sci. USA 1989. 86: 10049. 13 Osame, M., Igata, A. and Matsumoto, M., in Roman, G. C., Vernant, J. C. and Osame, M. (Eds.), HTLV-Iand the Nervous System, Alan R. Liss, Inc., New York 1989, p. 213. 14 Cairns, J. S., Curtsinger, J. M., Dahl, C. A., Freeman, S., Alter, B. J. and Bach, F. H., Nature 1985. 327: 166. 15 Gregersen, F! K., Shen, M., Song, Q.-L., Merryman, P., Degar, S., Seki,T., Maccari, J., Goldberg, D., Murphy, H., Schwenzer, J., Wang, C.Y., Winchester, R. J., Nepom, G. T. and Silver, J., Proc. Natl. Acad. Sci. USA 1986. 83: 2642. 16 Merryman, P., Gregersen, P. K., Lee, S., Silver, J., NunezRoldan, A., Crapper, R. and Winchester, R., J. Immunol. 1988. 140: 2447. 17 Gorski, J. and Mach, B., Nature 1986. 322: 67. 18 Gorski. J., Hum. lmmunol. 1989. 24: 145. 19 Geever, R. F., Wilson, L. B., Nallaseth, F. S., Milner, P. F., Bittner, M. and Wilson, J.T., Proc. Natl. Acad. Sci. USA 1981. 78: 5081. 20 Aizawa, M. (Ed.), HLA in Asia-Oceania 1986, Hokkaido Univ. Press, Sapporo 1986. 21 Sette, A , , Buus, S., Colon, S., Smith, J. A., Miles, C. and Grey, H. M., Nature 1987. 328: 395. 22 Ijichi, S., Eiraku, N., Osame, M., Izumo, S., Kubota, R., Maruyama, I., Matsumoto, M., Niimura, T. and Sonoda, S.. J. Neuroimmunol. 1989. 25: 251. 23 Brown, J. H., Jardetzky, T., Saper, M. A., Samraoui, B., Bjorkman, €? J. and Wiley, D. C., Nature 1988. 332: 845. 24 Goronzy, J., Weyand, C. M. and Farthman, G . C., J. Clin. Invest. 1986. 77: 1042. 25 Lombardi, G., Sidhu, S., Batchelor, J. R. and Lechler, R. I., Proc. Natl. Acad. Sci. USA 1989. 86: 4190.
Analysis of HLA-DR p l chain in HAM
1603
Short paper Koichiro UsukuO, Masatoyo NishizawaO, Kazumasa MatsukP, Katsushi TokunagaO, Keikichi TakahashiO, Nobutaka Eiraku., Masahito S u e h a d , Takeo JujiO, Mitsuhiro Osame. and Takeshi TabiraO Division of Demyelinating Disease and Agingo, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Blood Transfusion Servicen, Tokyo University Hospital, Tokyo and Third Department of Internal Medicine., Kagoshima University, Kagoshima
Association of a particular amino acid sequence of the HLA-DR p l chain with HTLV-I-associated myelopathy" Analyses of HLA-DRB1 gene using polymerase chain reaction and sequencespecific oligonucleotide probes reveal that the amino acid sequence Glu-Gln-Arg-Arg-Ala-Ala-Val at positions 69-75 of the third hypervariable region (HVR) of HLA-DR p l chain is significantly associated with HTLVI-associated myelopathy (HAM). Since the frequency of this sequence in HTLV-I carriers is almost the same as that in controls from Kagoshima (an endemic area of HTLV-I), this sequence may be related to susceptibility to HAM rather than to susceptibility to HTLV-I infection. Since this third HVR functions as putative antigen-binding sites and T cell recognition sites, the amino acid sequence of positions 68-73 was analyzed in detail. The analysis reveals that Gln'O and Arg'l are relevant to the occurrence of HAM.
1 Introduction Human Tcell lymphotropic virus type I (HTLV-I) causes at least two diseases, HTLV-I-associated myelopathy (HAM) [ 1]/HTLV-I-associated tropical spastic paraparesis (HTLVI/TSP) [2] and adult Tcell leukemia (ATL) [3]. The viruses isolated from patients with HAM and ATL are identical by the restriction enzyme analysis [4, 51 and by the nucleotide sequence analysis [6]. However, it is not known how HTLV-I induces two different diseases. Our previous study has demonstrated that HAM and ATL are associated with different HLA haplotypes; the frequency of HLA-DR1 and -DR4 is higher in HAM and the frequency of HLA-A26 is higher in ATL [7]. A strong association between acute type of ATL and HLA-Bw62Cw3-A26 haplotype is reported [8]. Furthermore, in HAM patients, a higher immune response to HTLV-I is demonstrated than in ATL patients [7]. These data suggest that HAM and acute type of ATL occur in two different populations, and that HAM is related to HLA-DR and ATL to HLA class I. Recent advances in technology of HLA typing make it possible to examine disease susceptibility through determi-
nation of specific amino acid (aa) sequence(s) [9-121, which may be shared among several alleles. In this report, we have focused on the p-chain sequence of the DR molecule to investigate which part of this chain is important for the occurrence of HAM. For this purpose, a specific segment of the HLA-DRB1 gene in human genomic DNA was amplified using polymerase chain reaction (PCR) and the sequence variation was analyzed with sequence-specific oligonucleotide (SSO) probes. We show a definite association of a particular D R p l chain sequence with HAM,which contributes to HAM susceptibility.
2 Materials and methods 2.1 Patients and control subjects For this study 32 patients with HAM, 8 patients with ATL, 46 asymptomatic HTLV-I carriers, 40 unrelated controls residing in Kagoshima, a prefecture in the southwestern Japan where HAM and ATL are prevalent [13], and 36 unrelated controls residing inTokyo, a non-endemic area of HTLV-I infection, were examined. All Tokyo controls were previously HLA typed. 2.2 Oligonucleotide primers and SSO probes
[I 81131
*
This work was supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture of Japan.
Correspondence: Masatoyo Nishizawa, Division of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, Japan Abbreviations: HTLV-I: Human T cell lymphotropic virus type I HAM: HTLV-I-associated myelopathy TSP: Tropical spastic paraparesis ATL: Adult Tcell leukemia PCR: Polymerase chain reaction SSO: Sequence-specific oligonucleotide aa: Amino acid(s) HVR: Hypervariable region 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
The DRBl oligonucleotide primers for PCR were GLPDRBl (5'-ITCTTCAATGGGACGGAGCG-3') and GAMPDRBl (5'-GCCGCTGCACTGTGAAGCTCTC3') which anneal t o the region of exon 2 of the DRB gene encoding the aa 17 to 23 and aa 87-94, respectively (Fig. 1). SSO probes have been designed to investigate specific aa sequences on HLA-DR p l chain in this study. Table 1 summarizes the nucleotide sequence of each probe, the aa position and sequence encoded by each probe and HLA alleles reported to include each aa sequence. To ensure the specificityof each probe, serologically typed standard DNA samples were amplified, slot blotted and hybridized simultaneously with test samples, and each spot on the blot was determined as positive or negative. 0014-2980/90/0707-1603$03.50+ .25/0
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K. Usuku, M. Nishizawa, K. Matsuki et al.
Table 1. Synthesized oligonucleotide probes
Probe 1 2 3 4 5C) 6
7 8
Oligonucleotide Sequencea)
aa sequence
THb)
T AC T T C T A T CA C CAAGAGGA CGGCCTAGCGCCGAGTAC GCAGAGGCGGGCCGCGGT CTGGAGCAGAGGCGGGCC
3UyFYHQEE36 "RPSAEY 6YEQRRAAV5 68LEQRRA73
48
. . . .
. . . .
. . . .
. . . .
.AG.C . . .AG.CGA .AG.C . . . . .G . . .
. . .C . . . . . . . . . .
("C)
68LEDRRA73 68LEDERA73 68LEDRRA73 6BLERRRA73
. . . . . . . . . . . . . .
63 70
66 64 63
64 73
Reported HLA allele DR4 DR4Dwl5,DRwS DR1,DR4Dwl4/15,DRwl4Dwl6 DR1,DR4Dw13/14/15/KT2, DRw14Dw16 DRw15DwU12 DR4DwlO,DRwl3 DRSDw5,DRwS DRw14Dw9,DRw9,DRw10
a) Each nucleotide sequence is written in order of 5'- to 3'-end. b) TH, temperature ("C) for hybridization. c) Periods indicate identity of the nucleotide to that of probe 4.
10
20
W1 CW4M4 W4Dw15 DRwl4Dwl6........EY CW4Dwl3 DR4DKT2
........ E. V.H....... ........E. V.H....... STS....... ........ E. V.H....... ........E. V.H....... ........Q. D.Y.......
DliWl6Dw2 DFMl6Dwl2........Q.
D.Y.......
R
40
..... F.D.Y .....F.D.Y
F.H...Y... F.H...Y... F...Y FH.... N... .....F. D.Y F.H...Y... F.D.Y F.H.......
..... .....
.....F.H.D ......DL.. ..... F.H.G ......N...
SIS....... Y....
SIS....... D......... V.........
.....F.D.Y FH.... Y... ..... Y.H.G ......N... ......... R VH.... YA.Y
m
........EY
DRw8
........EY
50
w IyNgEEsvRFV-
SIX..
........ E. V.H....... ........EY STS....... ...
E
.....FF..DD..YY ..... .....FF..DD..YY .....
lX4DwlO IXwl3
DRw14Dw9 ...EY DRw9 Q....K. CWwlO ........EE.
30 l
F.H...Y... FH.... N... F.....Y... F.....Y...
60 70 80 !TECGRPDAEY WSXFDLLEQ RR?AVtYlYG?
90 UBR
............................................. V.... .... ................ S... .................................. ...................................................... ................................. EE............ ..... ................................. .....VV........ .... .......................... FF....DD ........................ .......................... .................... .......................... I..D E......... .....V.... .... ......F... ................I..D E.,....... .....V.... .... ......F... .......E.. ......F..D ........................ ................ S... ......F..D ...L...... ...... ................A.DD .........R ...E...... .....V.... .... ................ V..S ......F..R ...E...V.. ...... ............................. R ........................
Figure 1. aa sequences (in one-letter code) of the first domain of the HLA-DR p l chain.The numbers above sequence refer to positions of the aa. Periods indicate identity with the DR1 sequence. Blanks indicate that no sequence information is available. Arrows indicate positionsof PCR primers. References of the sequences are as follows: DR4Dw13 from Cairns et al. [14];DR4Dw15 from Gregersen et al. [15]; DRwlOfrom Merryman et al. [16]; DRwl4Dw9 from Gorski and Mach [17]; DRw14Dw16from Gorski [18]; the others fromTodd et al. [9].
2.3 In vitro DNA amplification
2.4 Dot blotting and detection of specific sequence
Genomic DNA was extracted from the white blood cells of patients and controls as described previously [19]. PCR was carried out in 100 pl of the reaction mixture containing 1-5 pg genomic DNA, 10 mM Tris-HC1, pH 8.3, 50 mM KCI, 1.5 mM MgC12, 200 p~ of dATP, dlTP, dGTP and dCTP, and 1 pg of each primer. The sample was heated at 95 "C for 10 min, cooled to 50°C and 2.5 units of Taq DNA polymerase was (Perkin Elmer Cetus Corp., Norwalk, CT), then subjected to a thermal cycle with 72 "C for 2 min, 95 "C for 1min and 50 "C for 2 min. This procedure was repeated 30 times, then followed by an additional incubation at 72 "C for 5 min to complete the extention. After completion of the amplification, 6 pl of the sample was electrophoresed to confirm the amplification.
The amplified DNA sample was mixed with 530 pl of 0.5 M NaOH and 25 mM EDTA, then 200 pl of this mixture was blotted onto nylon filter (Zeta-Probe Blotting Membranes, Bio-Rad, Richmond, CA). The hybridization using each probe was carried out at the temperature summarized in Table 1. Each filter was prehybridized in a bag containing 5 X SSPE (720 mM NaCl, 40 mM NaH2P04,4 mM EDTA, pH 7.4), 5 x Denhardt's solution (0.1%BSA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone) and 5% SDS for 1 h at the hybridization temperature. The SSO probe (10 pmol) was 3' end-labeled with [a-32P]dideoxyATP(= 111MBq), and purified through a Sephadex column (NAPS; Pharmacia, Uppsala, Sweden). After prehybridization the 32P-labeled probe was added to the prehybridization buffer, and the
Analysis of HLA-DR fJ1 chain in HAM
Eur. J. Immunol. 1990. 20: 1603-1606
filter was hybridized for additional 1 h. The filter was then washed once in 2 x SSPE/2.5% SDS for 10 min, and once in 2 X SSPE/1.0% SDS for 10 min at the hybridization temperature, and then exposed with an intensifying screen at - 8 5 O C f o r 6 h t o 2 4 h .
Probe 2
1605
Probe 3
2.5 Statistical analysis Statistical analysis was done using the x2 test or the Fisher’s exact test.
3 Results Since HLA-DR1 and DR4 were frequently seen in patients with HAM [7], we first analyzed HLA-DR1 and DR4 using SSO Probes 1 to 3 (Table 2). Representative blots are shown in Fig. 2. The analysis shows that frequency identified positive by probe 3 (HLA-DR1, DR4Dw14/15, DRw14Dw16) in HAM patients is significantly higher than in ATL patients (p < 0.04, relative risk 6.60), HTLV-I carriers (p < 0.05, relative risk 2.86), controls in Kagoshima (p < 0.02, relative risk 3.30) or controls in Tokyo (p < 0.00005, relative risk 10.0). It is also noted that controls in Kagoshima have a higher frequency than those in Tokyo (p < 0.05, relative risk 3.30). Since allelic specificity of HLA-DR haplotype in the third hypervariable region (HVR) of HLA-DR p l chain can be classified into five groups in case of the Japanese, five SSO probes have been designed to further examine the aa difference in detail among aa residues 68-73 (HLA-DRwl6Dw21, DR3, DR4Dw4 and DR7 are known to be extremely rare among the Japanese [20], and have, therefore, been omitted in this study). Table 3 summarizes that only probe 4 (HLA-DR1, DR4Dw13/14/15/KT2, DRw14Dw16) showes significantly higher frequency in HAM patients than in ATL patients (p < 0.05, relative risk 7.67), HTLV-I carriers (p < 0.01, relative risk 3.67) or
Figure2. Representative slot blot of 20 patients with HAM. A specific segment of the HLA-DRB1 gene was amplified by the PCR method and hybridizid with probe 2 and probe 3.The number written on the left of each spot is an identity number of each HAM patient.
Kagoshima controls (p < 0.01, relative risk 3.83). Other probes do not show significant positive or negative association with HAM.
4 Discussion Recent studies of specific HLA class I1 sequences in patients with some autoimmune diseases and controls have provided more informative markers for genetic susceptibility, and suggested new strategies for clarifying pathomechanisms of the diseases [9, 101. A T cell recognizes a foreign antigen through interactions among the TcR, processed antigen fragments and the polymorphic residues of
Table 2. Dot blot analysis of HAM patients, ATL patients, HTLV-I carriers and controls
HAM ATL HTLV-I carrier Kagoshima control Tokyo control
n
Probe 1
Probe 2
Probe 3=)
32 8
13 (40.6)b) 3 (37.5) 20 (43.4) 15 (37.5) 11 (30.6)
14 (43.8) 2 (25.0) 23 (50.0) 20 (50.0) 12 (33.3)
22 (68.8) 2 (25.0) 20 (43.5) 16 (40.0) 6 (16.7)
46 40 36
a) Probe 3: HAM vs. ATL, relative risk (RR) = 6.60, x2 = 3.444, p < 0.04; HAM V S . HTLV-I camer, RR = 2.86, x2 = 4.856, p < 0.05: HAM vs. Kagoshima control, RR = 3.30. x2 = 5.896, p < 0.02; HAM vs. Tokyo control, RR = 10.0, x2 = 18.97, p < O.ooOo5: Kagoshimacontrol vs.Tokyocontro1, RR = 3.30. x2 = 5.015, p < 0.05. b) Percentages are shown in parentheses.
Table 3. Dot blot analysis of aa sequence 68-73 of HLA-DR p l chain in patients with HAM, patients with ATL, HTLV-I carriers and controls a) Probe 4: HAM vs. ATL, relative risk (RR) = 7.67, x* = n Probe4a) Probe5 Probe6 Probe 7 Probe8 4.167, p < 0.05; HAM vs. HTLV-I carrier, RR = 3.67, HAM 32 23 (71.9)b) 7 (21.9) 4 (12.5) 7 (21.9) 12 (37.5) = 7.210, p < 0.01: HAM vs. ATL 8 2 (25.0) 3 (37.5) l(12.5) 3 (37.5) 3 (37.5) Kagoshima control, RR = 3.83, 8 (19.0) HTLV-I carrier 42 17 (40.5) 17 (40.5) 3 (7.1) x2 = 7.276, p < 0.01. 22 (52.4) 14 (35.0) 2 (5.0) Kagoshima control 40 16 (40.0) 5 (12.5) 18 (45.0) b) Percentages are shown in parentheses.
x2
1606
K. Usuku, M. Nishizawa, K. Matsuki et al.
HLA class I1 molecules [21]. HAM shows several abnormal immune reactions probably mediated by activated T cells [22]. It is our aim to elucidate genetic susceptibility to HAM, and to clarify HLA class I1 sequences which may play important roles in the occurrence of HAM, through determination of specific aa sequences rather than conventional serological allelic typing. The present study reveals that the aa sequence EQRRAAV at positions 69-75 of the HLA-DR p l chain identified by probe 3 is significantlyassociated with HAM. Although the frequency of this sequence in HAM is the highest, it is almost the same between HTLV-I carriers and Kagoshima controls (Table 2). This may indicate that the aa sequence EQRRAAV is related to susceptibility to HAM rather than to susceptibility to HTLV-I infection. The difference in frequencies of this sequence observed in HAM and ATL patients is attributable to the difference in their respective genetic background [7]. The difference of frequency of this sequence in Kagoshima controls and Tokyo controls may be related to the geographical preponderance of HAM in Kagoshima [13]. According to the model of class I1 molecule proposed by Brown et al. (231, aa residues at positions 70 and 71 are within the putative antigen-binding site of HLA-DR b l chain a-helix. Highly polymorphic regions of the a-helix include those functioning also as T cell recognition sites [lo, 241. In particular, the third HVR of the HLA-DR fi1 chain, aa positions 65-75, includes not only an antigenbinding site [23] but also a target of alloreactiveTcel1 clones [25].We then examined the aa sequences of positions 68-73 in detail, because difference in the aa sequence in this region is determined by positions 70 and 71. As shown in Table 3, frequency of the aa sequence LEQRRA is significantly higher in HAM patients than in ATL patients, HTLV-I carriers or Kagoshima controls. This may indicate that the aa residues Gln7" and Arg71 are important in the occurrence of HAM. Furthermore, we have not found any particular aa residues or sequences in this region that are negatively associated with HAM, contributing to resistance to HAM. The results of the aa sequence analysis in rheumatoid arthritis (RA), a typical autoimmune disease [lo-121, clearly show that the aa sequence in the third HVR of HLA-DR1 and of some of HLA-DR4 haplotypes has positive association with RA. It is of interest to know that HAM and RA have association with the same HLA class I1 aa sequence. Although HTLV-I infection is the cause of HAM, the pathomechanism is still obscure. Nevertheless, it is important to know that there is a relationship between the particular aa sequence of HLA-DR p1 chain and HAM, and this has been accomplished by investigating aa sequences among different HLA-DR alleles. Further analyses are essential to reveal antigen(s) and T cells contributing to the occurrence of HAM.
Eur. J. Immunol. 1990. 20: 1603-1606 Received, November 16, 1989; in revised form March 12, 1990.
5 References 1 Osame, M., Matsumoto, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H.,Tara, M. and Igata, A., Ann. Neurol. 1987. 21: 117. 2 Gessain, A., Barin, E , Vernant, J. C., Gout, O., Maurs, L., Calender, A. and deThe, G . , Lancet 1985. ii: 407. 3 Yoshida, M., Miyoshi, I. and Hinuma,Y., Proc. Natl. Acad. Sci. USA 1982. 79: 2031. 4 Yoshida, M., Osame, M., Usuku, K.. Matsumoto, M. and Igata, A., Lancet 1987. i: 1085. 5 Imamura, J.,Tsujimoto, A., Ohta,Y., Hirose, S., Shimotohno, K., Miwa, M. and Miyoshi, I., Int. J. Cancer. 1988. 42: 221. 6 Tsujimoto, A., Terauchi, T., Imamura, J., Shimotohno, K., Miyoshi, 1. and Miwa. M., Mol. Biol. Med. 1988. 5: 29. 7 Usuku, K., Sonoda, S., Osame, M.,Yashiki, S.,Takahashi, K., Matsumoto, M., Sawada,T.,Tsuji, K.,Tara, M. and Igata, A., Ann. Neurol. 1988. 23: 143. 8 Uno, H., Kawano, K., Matsuoka, H. and Tsuda, K., Clin. Exp. Immunol. 1988. 71: 211. 9 Todd, J. A., Bell, J. I. and McDevitt, H. O., Nature 1987. 329: 599. 10 Todd, J. A., Acha-Orbea, H., Bell, J. I., Chao, N., Fronek, Z., Jacob, C. O., McDermott, M., Sinha, A. A.,Timmerman, L., Steinman, L. and McDevitt, H. O., Science 1988. 240: 1003. 11 Watanabe,Y.,Tokunaga, K., Matsuki, K.,Takeuchi, F., Matsuta, K., Maeda, H., Omoto, K. and Juji,T., J. Exp. Med. 1989. 169: 2263. 12 Wordsworth, B. P., Lanchbury, J. S. S., Sakkas, L. I.,Welsh, K. I., Panayi, G. S. and Bell, J. I., Proc. Natl. Acad. Sci. USA 1989. 86: 10049. 13 Osame, M., Igata, A. and Matsumoto, M., in Roman, G. C., Vernant, J. C. and Osame, M. (Eds.), HTLV-Iand the Nervous System, Alan R. Liss, Inc., New York 1989, p. 213. 14 Cairns, J. S., Curtsinger, J. M., Dahl, C. A., Freeman, S., Alter, B. J. and Bach, F. H., Nature 1985. 327: 166. 15 Gregersen, F! K., Shen, M., Song, Q.-L., Merryman, P., Degar, S., Seki,T., Maccari, J., Goldberg, D., Murphy, H., Schwenzer, J., Wang, C.Y., Winchester, R. J., Nepom, G. T. and Silver, J., Proc. Natl. Acad. Sci. USA 1986. 83: 2642. 16 Merryman, P., Gregersen, P. K., Lee, S., Silver, J., NunezRoldan, A., Crapper, R. and Winchester, R., J. Immunol. 1988. 140: 2447. 17 Gorski, J. and Mach, B., Nature 1986. 322: 67. 18 Gorski. J., Hum. lmmunol. 1989. 24: 145. 19 Geever, R. F., Wilson, L. B., Nallaseth, F. S., Milner, P. F., Bittner, M. and Wilson, J.T., Proc. Natl. Acad. Sci. USA 1981. 78: 5081. 20 Aizawa, M. (Ed.), HLA in Asia-Oceania 1986, Hokkaido Univ. Press, Sapporo 1986. 21 Sette, A , , Buus, S., Colon, S., Smith, J. A., Miles, C. and Grey, H. M., Nature 1987. 328: 395. 22 Ijichi, S., Eiraku, N., Osame, M., Izumo, S., Kubota, R., Maruyama, I., Matsumoto, M., Niimura, T. and Sonoda, S.. J. Neuroimmunol. 1989. 25: 251. 23 Brown, J. H., Jardetzky, T., Saper, M. A., Samraoui, B., Bjorkman, €? J. and Wiley, D. C., Nature 1988. 332: 845. 24 Goronzy, J., Weyand, C. M. and Farthman, G . C., J. Clin. Invest. 1986. 77: 1042. 25 Lombardi, G., Sidhu, S., Batchelor, J. R. and Lechler, R. I., Proc. Natl. Acad. Sci. USA 1989. 86: 4190.
