European Journal of Immunogenetics (1992), 19,425-430
SHORT COMMUNICATION A NEW R E S T R I C T I O N F R A G M E N T L E N G T H POLYMORPHISM OF THE H U M A N TNF-B G E N E DETECTED BY A s p H 1 DIGEST S . F E R E N C I KM, .* L I N D E M A N NB, .* H O R S T H E M K E& ,?H. G R O S S E - W I L D E *
*Department of Immunology, and +Department of Human Genetics, University Hospital of Essen, Medical School, Germany (Received 27 July 1992; accepted 5 August 1992)
Tumour necrosis factor-alpha (TNF-alpha) and tumour necrosis factor-beta (TNF-beta) play an important role in the immune response. The cytokines TNF-(Uand TNF-P are polypeptides and signal molecules, secreted by activated macrophages (Nedwin et al., 1985) and stimulated lymphocytes (Aggarwal et al., 1985). Their structural genes (TNF-A and TNF-B) are located within the human major histocompatibility complex (MHC) between HLA-B and C2 (complement component C2) on chromosome 6p and comprise a region of about 3 kilobases (kb). In 1986 Nedospasov et al. sequenced both TNF genes and reported only a low degree of sequence variations. For TNF-B, due to a variable site within the first intron a restriction fragment length polymorphism (RFLP) using the endonuclease NcoI, has been described (Webb et a l . , 1990) which is strongly associated to the HLA-B8 allele (Fugger et al., 1989) and a rare restriction site for EcoRI (Partanen etal., 1988). As a consequence of variable methylation at AccI sites within and upstream of the TNF-B gene an additional non-allelic polymorphism was reported (Webb et a l . , 1990). There were other restriction endonucleases studied but they did not yield a genomic TNF polymorphism (Fugger et al., 1989). Furthermore there exist three ‘microsatellite’ regions (TNF a, TNF b, and TNF c) within a 12kb region of the human MHC that includes the TNF loci (Jongeneel et al., 1991). Whereas TNF c is located within the first intron of the TNF-B gene, TNF a and b are approximately 3.5 kb telomeric apart from TNF-B. These ‘microsatellite’ regions are polymorphic regions with 13, 7, and 2 alleles, respectively. In a search for further RFLP in the TNF-B region we used the following 15 endonucleases: AspI, AspHI, Asp700, AvaII, BanI, BanII, BclI, BglI, DraI, H i n f I , HpaI, M v a I , NciI, Sau961, and S t u l . The TNF-B probe was a 2.4kb EcoRI fragment incorporated into plasmid pST18 (Dunn et a l . , 1983). High molecular weight human DNA was extracted from Epstein-Barr virus-transformed B-lymphoblastoid cell lines by the salting out procedure as described by Miller et al. (1988). The endonucleases digested 15 pg of DNA (Boehringer Mannheim, Germany) with 5 U Kg-‘ DNA for 18h at 37°C and the fragments were monitored on minigels. After ethanol precipitation the Correspondence: Dr H. Grosse-Wilde, lnstitut fur Immunologie, Universitatsklinikum Essen, Virchowstrasse 17 1, D-4300 Essen 1, Germany. 425 C
426
S. Fercncik et al.
genomic D N A was fractionated by electrophoresis in 0.7% SeaKem-agarose gel (FMC BioProducts Rockland, USA) with 0 . 0 4 Tris-acetate ~ and 2 m E~D T A , p H 7.8 for 20h at 2OV. Southern-blotting was carried out for 16h using 20 X SSC (3M NaCl, 1 . 5 sodium ~ citrate) by capillary transfer as described by Maniatis et al. (1982) with a Biodyne A-membrane (Pall BioSupport Portsmouth, UK). The membrane was dried at 80°C for 1h. The prehybridization was performed in a mixture consisting of 50% formamide, 5”/0 SSC, 0.02% SDS and 0.1% N-Laurylsarkosin. The nylon membranes were shaked for 24h at 40°C in a water bath. The hybridization with a digoxigenin random-priming labelled TNF-B cDNA (20ng ml- hybridization mixture) was performed 20h at 40°C under slow shaking. Washing of membranes and colorimetric detection were done according to the protocol of Boehringer Mannheim. For a lower background we used 0.5% blocking reagent plus 0.5% casein. After a first screening step of 10 different H L A homozygous cell lines marked by an asterisk in Table 1, only Asp HI (recognition sequence G(A/T)GC(AIT)’C) showed a polymorphism with fragments of 10 and/or 2 plus Skb, where the latter bands appeared always together. Using a TNF-A probe (2.75kb EcoRI fragment in plasmid pST18) no RFLP for AspHI could be detected. The genomic structure of TNF-B is given in Fig. 1. Bright boxes are the untranslated, dark boxes the translated regions. In the map described by Nedospasov et al. (1986) a restriction site at position 3106/7 can be found for AspHI (GAGCT‘C). Assuming a base exchange G K in the first intron of TNF-B at position 1184/5 (GTGCTG) one obtains GTGCT‘C instead of this published sequence and thereby a further restriction site for AspHI, resulting in the observed 2 and 8 k b fragments instead of the lOkb fragment. Our assumption is supported by Messer et al.
’
TABLE 1. TNF-B specific restriction fragment sizes (kb) detected by AspHI digest in 74 HLA typed reference cell lines
HLA
I 2 3 4 5 6 7 8
9 10 11
12 13 14 15
16 17 18 19 20 21
ECBFUWS No.
Local ID
A
Cw
10W9002 10W9005 E20103901 10W9006 E21808103 E21931102 E29928803 09w0101 10W9003* 09W0102 E20146901 10W9001 10W9013* 10W9017 low9082 10W9014 10W9008 10W9011 10W9010 low9015 10W9084
MZ070782 HOM2 FEE WTIOOBIS FUH THC WAM AL KAS116 HEN VEH SA SCHU WT8 H0104 MGAR DO208915 E4181324 AMAI WT24 Calogero
24 3 2 11 2,3 3 3, 11 3 24 2,29 2 24 3 3 3 26 25
2, 8 1
1
28 2 2
1
4 4 4 4 4 4 1 7 7 7 7 7 4 2 2
TNF-B B
10
14 27 27 35 35 35 35 35.51 51 44 56 7 7 7 7 8
x x x
x x x x x
x x
X
x
X X
HLA DR
DRBI* 0102 0101 0l0x 0101
5 5 1
Ol0X 0l0x
1
0l0x 0101
1 1 1 1
1 1 1 1 1
X
X
15 16
X
16
X X
X X
x
DQ
1 1 1 1 1 1
15 15 15 15 15 15
X
18
52 53 27 61
2+8
0101 0101 0l0x 0101 1501 1501
1501 1501 1501 1502 1503 1601 160x
5
5 1 1 1 6 1 6 6 6 6 h
5 5
Asp HI digest and the human TNF-B gene HLA
22 23 24 2s 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
52 53 54 55 56 57 58 59 60 61
62 63 64 65 66 67 68 69 70 71 72 73 74
ECBWWS No.
Local ID
low9022 10W9023* 09W0301 low9088 low9087 10w9019 10W9018 10W9021 10W9091 10W9031' 10~9032 10W9033 10W9034 10W9026 09W1303 low9098 IOW9024 09W1004 low9043 10W9044 10W9039 10W9040 10W9042' 10W9038' 10W9065 10W9060 low9055 low9053 low9063 09W0604 10W9059" 10W9097 09W0607 E20146801 09W1802 low9056 10w9057 10W9061 10WW94 09w1102 10W9049 low9051 lOW9048" 10W9046 10W9047 10W9068* low9066 10W9070 10w9071 low9073 10W9075* low9074 E20160101
COX VAVY AVL PF040 15 STEINLIN DUCAF Lon8 1785 RSH MLF BOLETH BSM BM14 SAVC YAR SSTO MTI4B KT17 RLO BM21 BRIP J VM BM15 TISI BM16 HHKB CB6B HO301 HOR WT47 SCL SLEOUS EMJ KRA REB HAG 1 KOSE TEM 3 1227ABO CF996 GRO IBW9 PITOUT LBUF BH13 PLH B M9 TAB089 LUY OLGA KT12 DKB HID RAJI
A
Cw
I 1 1
I 1
30 2, 24 30,68 2 2 3 3 3 26 32 31 2, 11 3, 26 1 24 2 1 24 2 3 1 3 33 32 2 2 2, 3 2 2 2 2 26 2 2. 3 2 33 29 30 2 3 2 2 2 31 24,31 24 2 3
TNF-B B
7 7 7 7 5 5 2 9 10
7 7 7 -
5 10 4,9 5 7 4 7 7 9 8
5 5 10 10
3 3 7 7 8 6 8 4 6 6 6 4 -
4 10 8, 10 3.4
10 8 8 8 8
x x x x
8
x x
18 18 42 62 62 62 7 7 38 44 60 35,62 38,41 41 51,63 18
49 35 18 7 62 14 44 44 44 60 60 60 60 41 35 38 18 14 57 65 44 13 13 47 35 46 51 62 35,52 60 60,61 35.62
2+8
x x x x x X X X
x X
x x
X
X
x x x
X
X
x x
12 X
x x x x x x x x
X
x x X X X
x x x X X
X X
x x x x x x x x
427 HLA
DR 17 17 17 17 17 17 18 18 4 4 4 4 4 4 4
DRBI*
0301 0301 0301 0301 030 1 0301 0302 0302 040 1 0401 040 I 0401 0401 0402 0403 4 0404 4 0406 4 040x 11 1101 11 1lox 11 1102 II 1102 I 103 11 1201 7 13 1301 13 1301 6 1302 1302 13 13 1302 13 I30x 13 1302 13 1302 13 1302 6 130x 13 1303 1 x 1 4 1302, 1401 14 1401 14 1401 7 070x 7 070x 7 070 1 7 0701 7 0702 7 0702 7 0702 8 080 1 8 0803 8 0803 8 0802 9 090 1 9 090 1 9 090 1 10. 17 0301. 1001
DQ 2 2 2 2 2 2 2 4 7 8 8 8 8 8 3 8
8 3 7 7 7 7 7 6
5 6 6
6 1 6 6 1 1 1 5 1
5 2 2 2 2 2 2 2 4 6 7 4 9 9 9 2
428
S . Ferencik et al. TNF-B
FIG.1 . Genornicstructure of the TNF-B gene region and restriction sites for Asp HI based on the data of Nedospasov ef al. (1986) and this study.
(1991) reporting recently a nucleotide exchange at a corresponding position after sequencing the 5' part of another TNF-B gene. A further telomeric restriction site not analysed by Messer et ul. must be postulated to explain the existence of the 8 kb fragment observed. The AspHI polymorphism was then studied in 74 HLA typed reference cell lines from different International Histocompatibility Workshops and the ECBR cell bank (Grosse-Wilde & Ferrara, 1992). The results are summarized in Table 1. The overall RFLP results for TNF-B based on AspHI digest show an allele frequency of 0.66 for the lOkb fragment and of 0.34 for the 2+8kb fragments. Thus, compared to the reported NcoI polymorphism of TNF-B with 10.5 kb and 5.1 + 5.4kb bands having a frequency of 0.71 and 0.29 respectively (Fugger et ul., 1989), the AspHI polymorphism is more informative. Based on the data of Badenhoop et al. (1990) analysing 48 of the 74 cell lines used in this study there was no correlation between the NcoI and the AspHI polymorphisms at TNF-B. This observation is at variance to the data of Messer et al. (1991) analysing 14 individuals. From that one must conclude that NcoI and AspHI restriction sites should be mutually exclusive. This Asp HI restriction site is located in the first intron of TNF-B as the position of the microsatellite TNF c. The distance between both is 53bp. So the first intron of TNF-B appears to be the most polymorphic region of this gene. With regard to the allelic association between the AspHI RFLP at the TNF-B locus and the alleles of the adjacent HLA loci, there was a statistically significant correlation ( P = 0.001) between the presence of the 2+8kb bands and the HLA-B7 allele irrespective of the HLA-DR types of the cells. An associztion to B7 and DR2 was found for microsatellite TNF a allele 11 by Jongeneel etal. (1991). For HLA-DR the most prominent findings are, that nearly all HLA-DR1, 3,8, and 9 positive cells carry the lOkb fragment, whereas HLA-DR2 seems to be associated with the 2+8kb fragments of TNF-B. Here, the exception is the 10W9010 (AMAI) cell typed during the 11th International Histocompatibility Workshop as HLA-DRB1*1503 (Kimura et a l . , 1992). The HLA-DR4, 5 , 6, and 7 positive cells displayed an heterogeneous picture for the AspI-II RFLP. To demonstrate the inheritance of this new TNF-B genomic polymorphism we analysed a family as given in Fig. 2, where the father (F) possesses the 2+8 kb Asp HI RFLP. As can be seen in the lower part of Fig. 2, these bands are transmitted to C1, C2, and C4 in coupling with the HLA haplotype a.
-
Asp HI digest and the human TNF-B gene
kb
MI1
F
M
c1
c2
c3
429
c4
23.13
9.42
10.0
8.0
6.56
2.32
2.03
2.0
FIG.2. Segregation analysis o f AspHI induced RFLP of TNF-B. The parental HLA haplotypes are: a A2,B44,Cw4,DR7,DQ2, b A2,B3S,Cw4,DRl ,DQI , c A2,B58,Cw7, DR l , DQI , d A28,B64,Cw8,DR5(12),DQ3.The TNF-€3 specific 2 + 8 k b bands are in coupling with haplotype a. MI1 is the molecular weight marker.
In summary, after a systematic analysis of 15 different endonucleases using a TNF-B specific cDNA probe amongst 74 immunogenetically defined reference cell lines we could define a new RFLP for this immunobiologically important gene within the human MHC which is supported by the recent TNF-B sequence data of Messer eta!. (1991). Compared to the well known AccI, EcoRI or NcoI based RFLP for TNF-B the use of AspHI beside microsatellite typing appears to be informative allowing further immunogenetic studies in intra-HLA recombinant families as well as patients with HLA linked or associated diseases.
430
S. Ferencik e t a1. ACKNOWLEDGMENTS
The study was supported in part by the Deutsche Forschungsgemeinschaft, G r 608/5-1. We thank Ms D r E. Weiss, Institut fur Immunologie, LMU Miinchen, Germany for the TNF-A and TNF-B cDNA probes. This article is a partial fulfilment of requirements for the doctor’s degree at the Medical Faculty, University of Essen, for Ms M. Lindemann.
REFERENCES ACGARWAL, B.B., EESSALU, T.E. & HAS, P.E. (1985) Characterization of receptors for human tumor necrosis factor and their regulation by gamma interferon. Nature, 318, 665. P., TROWSDALE, J., USADEL,K.H., GALE,E.A.M. & BOTTAZZO, BADENHOOP, K., SCHWARZ, G., BINGLEY, G.F. (1990) TNF-alpha gene polymorphisms: Association with type 1 (insulin dependent) diabetes mellitus. Journal of Immunogenetics, 16, 455. F.W. (1983) Complete Nucleotide Sequence of Bacteriophage T7 DNA and the DUNN,J.J. & STUDIER, Locations of T7 Genetic Elements. Journal of Molecular Biology, 166,477. FUGCER, L., MORLINC, N., RYDER,L.P., PLATZ,P., GEORGSEN, J., JAKOBSEN,B.K., SVEJGAARD, A. DALHOFF, K. & RANER, L. (1989) Ncol restriction fragment length polymorphism (RFLP) of the tumor necrosis factor (TNF alpha) region in primary biliary cirrhosis and in healthy danes. Scandinavian Journal of ImmunoLogy, 30, 185. GROSSE-WILDE, H. & FERRARA, G.B. (1992) European Collection for Biomedical Research. European Journal of Immunogenetics, 19, 181. JONCENEEL, C.V., BRIANT, L., UDALOVA, I.A., SEVIN, A., NEDOSPASOV,S.A. & CAMBON-THOMSEN, A. (1991) Extensive genetic polymorphism in the human tumor necrosis factor region and relation to extended HLA haplotypes. Proceedings of the National Academy of Science, USA, 88, 9717. KIMURA,A , , DONG,R.-P., HARADA, H. & SASAZUKI, T. (1992) DNA typing of HLA Class I1 genes in B-lymphoblastoid cell lines homozygous for HLA. Tissue Antigens, 40, 5. MANIATIS, T., FRITSCH, E.F. & SAMBROOK, J . (1982) Molecular Cloning: Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 382. MESSER,G., SPENGLER, U., JUNC, M.C., HONOLD, G., BLOMER, K., PAPE,G.R., RIETHMULLER, G. & WEISS,E.H. (1991) Polymorphic structure of the tumor necrosis factor (TNF) locus: An NcoI polymorphism in the first intron of the human TNF-P gene correlates with a variant amino acid in position 26 and a reduced level of TNF-P production. Journal of Experimental Medicine, 1763, 209. MILLER, S.A., DYKES,D.D. & POLESKY, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16, 1215. NEDOSPASOV,S.A., SHAKHOV, A.N., TURETSKAYA, R.L., METT,V.A., Azizov, M.M., GEORGIEV, G.P., KOROBKO,V.G. DOBRYNIN, V.N., FILLIPOV, S.A., BYSTROV, N.S., BOLDYREVA, E.I., CHUVPILO, S.A., CHUMAROV, A.M., SHINGAROVA, L.N. & OVCHINNIKOV, Y.A. (1986) Tandem arrangement of genes coding for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) in the human genome. Cold Spring Harbor Symposia on Quantitative Biology, 51,61 I . NEDWIN, G.E., NAYLOR, S.L., SAKAGUCHL, A.Y., SMITH,D., JARRETT-NEDWIN, J., PENNICA, D., GOEDDEL, D.V. & GRAY,P.W. (1985) Human lymphotoxin and tumor necrosis factor genes. Nucleic Acids Research, 13, 6361. PARTANEN, J. & KOSKIMIES, S. (1988) Low degree of DNA polymorphism in the HLA-linked lymphotoxin (tumor necrosis factor p) gene. Scandinavian Journal of Immunology, 28,313. WEBB,G.C. & CHAPLIN, D.D. (1990) Genetic variability at the human tumor necrosis factor loci. The Journal of Immunology, 145, 1278.
SHORT COMMUNICATION A NEW R E S T R I C T I O N F R A G M E N T L E N G T H POLYMORPHISM OF THE H U M A N TNF-B G E N E DETECTED BY A s p H 1 DIGEST S . F E R E N C I KM, .* L I N D E M A N NB, .* H O R S T H E M K E& ,?H. G R O S S E - W I L D E *
*Department of Immunology, and +Department of Human Genetics, University Hospital of Essen, Medical School, Germany (Received 27 July 1992; accepted 5 August 1992)
Tumour necrosis factor-alpha (TNF-alpha) and tumour necrosis factor-beta (TNF-beta) play an important role in the immune response. The cytokines TNF-(Uand TNF-P are polypeptides and signal molecules, secreted by activated macrophages (Nedwin et al., 1985) and stimulated lymphocytes (Aggarwal et al., 1985). Their structural genes (TNF-A and TNF-B) are located within the human major histocompatibility complex (MHC) between HLA-B and C2 (complement component C2) on chromosome 6p and comprise a region of about 3 kilobases (kb). In 1986 Nedospasov et al. sequenced both TNF genes and reported only a low degree of sequence variations. For TNF-B, due to a variable site within the first intron a restriction fragment length polymorphism (RFLP) using the endonuclease NcoI, has been described (Webb et a l . , 1990) which is strongly associated to the HLA-B8 allele (Fugger et al., 1989) and a rare restriction site for EcoRI (Partanen etal., 1988). As a consequence of variable methylation at AccI sites within and upstream of the TNF-B gene an additional non-allelic polymorphism was reported (Webb et a l . , 1990). There were other restriction endonucleases studied but they did not yield a genomic TNF polymorphism (Fugger et al., 1989). Furthermore there exist three ‘microsatellite’ regions (TNF a, TNF b, and TNF c) within a 12kb region of the human MHC that includes the TNF loci (Jongeneel et al., 1991). Whereas TNF c is located within the first intron of the TNF-B gene, TNF a and b are approximately 3.5 kb telomeric apart from TNF-B. These ‘microsatellite’ regions are polymorphic regions with 13, 7, and 2 alleles, respectively. In a search for further RFLP in the TNF-B region we used the following 15 endonucleases: AspI, AspHI, Asp700, AvaII, BanI, BanII, BclI, BglI, DraI, H i n f I , HpaI, M v a I , NciI, Sau961, and S t u l . The TNF-B probe was a 2.4kb EcoRI fragment incorporated into plasmid pST18 (Dunn et a l . , 1983). High molecular weight human DNA was extracted from Epstein-Barr virus-transformed B-lymphoblastoid cell lines by the salting out procedure as described by Miller et al. (1988). The endonucleases digested 15 pg of DNA (Boehringer Mannheim, Germany) with 5 U Kg-‘ DNA for 18h at 37°C and the fragments were monitored on minigels. After ethanol precipitation the Correspondence: Dr H. Grosse-Wilde, lnstitut fur Immunologie, Universitatsklinikum Essen, Virchowstrasse 17 1, D-4300 Essen 1, Germany. 425 C
426
S. Fercncik et al.
genomic D N A was fractionated by electrophoresis in 0.7% SeaKem-agarose gel (FMC BioProducts Rockland, USA) with 0 . 0 4 Tris-acetate ~ and 2 m E~D T A , p H 7.8 for 20h at 2OV. Southern-blotting was carried out for 16h using 20 X SSC (3M NaCl, 1 . 5 sodium ~ citrate) by capillary transfer as described by Maniatis et al. (1982) with a Biodyne A-membrane (Pall BioSupport Portsmouth, UK). The membrane was dried at 80°C for 1h. The prehybridization was performed in a mixture consisting of 50% formamide, 5”/0 SSC, 0.02% SDS and 0.1% N-Laurylsarkosin. The nylon membranes were shaked for 24h at 40°C in a water bath. The hybridization with a digoxigenin random-priming labelled TNF-B cDNA (20ng ml- hybridization mixture) was performed 20h at 40°C under slow shaking. Washing of membranes and colorimetric detection were done according to the protocol of Boehringer Mannheim. For a lower background we used 0.5% blocking reagent plus 0.5% casein. After a first screening step of 10 different H L A homozygous cell lines marked by an asterisk in Table 1, only Asp HI (recognition sequence G(A/T)GC(AIT)’C) showed a polymorphism with fragments of 10 and/or 2 plus Skb, where the latter bands appeared always together. Using a TNF-A probe (2.75kb EcoRI fragment in plasmid pST18) no RFLP for AspHI could be detected. The genomic structure of TNF-B is given in Fig. 1. Bright boxes are the untranslated, dark boxes the translated regions. In the map described by Nedospasov et al. (1986) a restriction site at position 3106/7 can be found for AspHI (GAGCT‘C). Assuming a base exchange G K in the first intron of TNF-B at position 1184/5 (GTGCTG) one obtains GTGCT‘C instead of this published sequence and thereby a further restriction site for AspHI, resulting in the observed 2 and 8 k b fragments instead of the lOkb fragment. Our assumption is supported by Messer et al.
’
TABLE 1. TNF-B specific restriction fragment sizes (kb) detected by AspHI digest in 74 HLA typed reference cell lines
HLA
I 2 3 4 5 6 7 8
9 10 11
12 13 14 15
16 17 18 19 20 21
ECBFUWS No.
Local ID
A
Cw
10W9002 10W9005 E20103901 10W9006 E21808103 E21931102 E29928803 09w0101 10W9003* 09W0102 E20146901 10W9001 10W9013* 10W9017 low9082 10W9014 10W9008 10W9011 10W9010 low9015 10W9084
MZ070782 HOM2 FEE WTIOOBIS FUH THC WAM AL KAS116 HEN VEH SA SCHU WT8 H0104 MGAR DO208915 E4181324 AMAI WT24 Calogero
24 3 2 11 2,3 3 3, 11 3 24 2,29 2 24 3 3 3 26 25
2, 8 1
1
28 2 2
1
4 4 4 4 4 4 1 7 7 7 7 7 4 2 2
TNF-B B
10
14 27 27 35 35 35 35 35.51 51 44 56 7 7 7 7 8
x x x
x x x x x
x x
X
x
X X
HLA DR
DRBI* 0102 0101 0l0x 0101
5 5 1
Ol0X 0l0x
1
0l0x 0101
1 1 1 1
1 1 1 1 1
X
X
15 16
X
16
X X
X X
x
DQ
1 1 1 1 1 1
15 15 15 15 15 15
X
18
52 53 27 61
2+8
0101 0101 0l0x 0101 1501 1501
1501 1501 1501 1502 1503 1601 160x
5
5 1 1 1 6 1 6 6 6 6 h
5 5
Asp HI digest and the human TNF-B gene HLA
22 23 24 2s 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
52 53 54 55 56 57 58 59 60 61
62 63 64 65 66 67 68 69 70 71 72 73 74
ECBWWS No.
Local ID
low9022 10W9023* 09W0301 low9088 low9087 10w9019 10W9018 10W9021 10W9091 10W9031' 10~9032 10W9033 10W9034 10W9026 09W1303 low9098 IOW9024 09W1004 low9043 10W9044 10W9039 10W9040 10W9042' 10W9038' 10W9065 10W9060 low9055 low9053 low9063 09W0604 10W9059" 10W9097 09W0607 E20146801 09W1802 low9056 10w9057 10W9061 10WW94 09w1102 10W9049 low9051 lOW9048" 10W9046 10W9047 10W9068* low9066 10W9070 10w9071 low9073 10W9075* low9074 E20160101
COX VAVY AVL PF040 15 STEINLIN DUCAF Lon8 1785 RSH MLF BOLETH BSM BM14 SAVC YAR SSTO MTI4B KT17 RLO BM21 BRIP J VM BM15 TISI BM16 HHKB CB6B HO301 HOR WT47 SCL SLEOUS EMJ KRA REB HAG 1 KOSE TEM 3 1227ABO CF996 GRO IBW9 PITOUT LBUF BH13 PLH B M9 TAB089 LUY OLGA KT12 DKB HID RAJI
A
Cw
I 1 1
I 1
30 2, 24 30,68 2 2 3 3 3 26 32 31 2, 11 3, 26 1 24 2 1 24 2 3 1 3 33 32 2 2 2, 3 2 2 2 2 26 2 2. 3 2 33 29 30 2 3 2 2 2 31 24,31 24 2 3
TNF-B B
7 7 7 7 5 5 2 9 10
7 7 7 -
5 10 4,9 5 7 4 7 7 9 8
5 5 10 10
3 3 7 7 8 6 8 4 6 6 6 4 -
4 10 8, 10 3.4
10 8 8 8 8
x x x x
8
x x
18 18 42 62 62 62 7 7 38 44 60 35,62 38,41 41 51,63 18
49 35 18 7 62 14 44 44 44 60 60 60 60 41 35 38 18 14 57 65 44 13 13 47 35 46 51 62 35,52 60 60,61 35.62
2+8
x x x x x X X X
x X
x x
X
X
x x x
X
X
x x
12 X
x x x x x x x x
X
x x X X X
x x x X X
X X
x x x x x x x x
427 HLA
DR 17 17 17 17 17 17 18 18 4 4 4 4 4 4 4
DRBI*
0301 0301 0301 0301 030 1 0301 0302 0302 040 1 0401 040 I 0401 0401 0402 0403 4 0404 4 0406 4 040x 11 1101 11 1lox 11 1102 II 1102 I 103 11 1201 7 13 1301 13 1301 6 1302 1302 13 13 1302 13 I30x 13 1302 13 1302 13 1302 6 130x 13 1303 1 x 1 4 1302, 1401 14 1401 14 1401 7 070x 7 070x 7 070 1 7 0701 7 0702 7 0702 7 0702 8 080 1 8 0803 8 0803 8 0802 9 090 1 9 090 1 9 090 1 10. 17 0301. 1001
DQ 2 2 2 2 2 2 2 4 7 8 8 8 8 8 3 8
8 3 7 7 7 7 7 6
5 6 6
6 1 6 6 1 1 1 5 1
5 2 2 2 2 2 2 2 4 6 7 4 9 9 9 2
428
S . Ferencik et al. TNF-B
FIG.1 . Genornicstructure of the TNF-B gene region and restriction sites for Asp HI based on the data of Nedospasov ef al. (1986) and this study.
(1991) reporting recently a nucleotide exchange at a corresponding position after sequencing the 5' part of another TNF-B gene. A further telomeric restriction site not analysed by Messer et ul. must be postulated to explain the existence of the 8 kb fragment observed. The AspHI polymorphism was then studied in 74 HLA typed reference cell lines from different International Histocompatibility Workshops and the ECBR cell bank (Grosse-Wilde & Ferrara, 1992). The results are summarized in Table 1. The overall RFLP results for TNF-B based on AspHI digest show an allele frequency of 0.66 for the lOkb fragment and of 0.34 for the 2+8kb fragments. Thus, compared to the reported NcoI polymorphism of TNF-B with 10.5 kb and 5.1 + 5.4kb bands having a frequency of 0.71 and 0.29 respectively (Fugger et ul., 1989), the AspHI polymorphism is more informative. Based on the data of Badenhoop et al. (1990) analysing 48 of the 74 cell lines used in this study there was no correlation between the NcoI and the AspHI polymorphisms at TNF-B. This observation is at variance to the data of Messer et al. (1991) analysing 14 individuals. From that one must conclude that NcoI and AspHI restriction sites should be mutually exclusive. This Asp HI restriction site is located in the first intron of TNF-B as the position of the microsatellite TNF c. The distance between both is 53bp. So the first intron of TNF-B appears to be the most polymorphic region of this gene. With regard to the allelic association between the AspHI RFLP at the TNF-B locus and the alleles of the adjacent HLA loci, there was a statistically significant correlation ( P = 0.001) between the presence of the 2+8kb bands and the HLA-B7 allele irrespective of the HLA-DR types of the cells. An associztion to B7 and DR2 was found for microsatellite TNF a allele 11 by Jongeneel etal. (1991). For HLA-DR the most prominent findings are, that nearly all HLA-DR1, 3,8, and 9 positive cells carry the lOkb fragment, whereas HLA-DR2 seems to be associated with the 2+8kb fragments of TNF-B. Here, the exception is the 10W9010 (AMAI) cell typed during the 11th International Histocompatibility Workshop as HLA-DRB1*1503 (Kimura et a l . , 1992). The HLA-DR4, 5 , 6, and 7 positive cells displayed an heterogeneous picture for the AspI-II RFLP. To demonstrate the inheritance of this new TNF-B genomic polymorphism we analysed a family as given in Fig. 2, where the father (F) possesses the 2+8 kb Asp HI RFLP. As can be seen in the lower part of Fig. 2, these bands are transmitted to C1, C2, and C4 in coupling with the HLA haplotype a.
-
Asp HI digest and the human TNF-B gene
kb
MI1
F
M
c1
c2
c3
429
c4
23.13
9.42
10.0
8.0
6.56
2.32
2.03
2.0
FIG.2. Segregation analysis o f AspHI induced RFLP of TNF-B. The parental HLA haplotypes are: a A2,B44,Cw4,DR7,DQ2, b A2,B3S,Cw4,DRl ,DQI , c A2,B58,Cw7, DR l , DQI , d A28,B64,Cw8,DR5(12),DQ3.The TNF-€3 specific 2 + 8 k b bands are in coupling with haplotype a. MI1 is the molecular weight marker.
In summary, after a systematic analysis of 15 different endonucleases using a TNF-B specific cDNA probe amongst 74 immunogenetically defined reference cell lines we could define a new RFLP for this immunobiologically important gene within the human MHC which is supported by the recent TNF-B sequence data of Messer eta!. (1991). Compared to the well known AccI, EcoRI or NcoI based RFLP for TNF-B the use of AspHI beside microsatellite typing appears to be informative allowing further immunogenetic studies in intra-HLA recombinant families as well as patients with HLA linked or associated diseases.
430
S. Ferencik e t a1. ACKNOWLEDGMENTS
The study was supported in part by the Deutsche Forschungsgemeinschaft, G r 608/5-1. We thank Ms D r E. Weiss, Institut fur Immunologie, LMU Miinchen, Germany for the TNF-A and TNF-B cDNA probes. This article is a partial fulfilment of requirements for the doctor’s degree at the Medical Faculty, University of Essen, for Ms M. Lindemann.
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