Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.
Since a dysregulated synthesis of tumor necrosis factor alpha (TNF-alpha) may be involved in the pathogenesis of autoimmune diseases, it was of interest to precisely locate the recently reported NcoI restriction fragment length polymorphism (RFLP) of the TNF-alpha region. However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic NcoI restriction site within the first intron of the TNF-beta gene and not in the TNF-alpha gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-alpha/TNF-beta production of phytohemagglutinin-stimulated peripheral blood mononuclear cells of individuals homozygous for the TNF-beta NcoI RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb NcoI fragment presented with a significantly higher TNF-beta response. A mRNA analysis demonstrated that higher protein levels of TNF-beta correlate also with increased amounts of TNF-beta transcripts. No allelic association was found in respect to TNF-alpha production. To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb of the 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-beta sequences. By computer-aided recognition motif search of DNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 of the TNF-beta gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 bp of the 5' part of TNF-beta of individuals typed homozygously for the NcoI RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB*2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-beta does not segregate with either of the two alleles. Thus, four TNFB alleles can be defined at the DNA level.
Messer
G
G
Institute of Immunology, Ludwig-Maximilians University, Munich 2, Federal Republic of Germany.
Spengler
U
U
Jung
M C
MC
Honold
G
G
Blömer
K
K
Pape
G R
GR
Riethmüller
G
G
Weiss
E H
EH
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008233:Lymphotoxin-alpha / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010835:Phytohemagglutinins / Q000494:pharmacology; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic / Q000235:genetics; D015183:Restriction Mapping; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D008233:Lymphotoxin-alpha; D010835:Phytohemagglutinins; D014409:Tumor Necrosis Factor-alpha; D015246:Deoxyribonuclease EcoRI; C052380:endodeoxyribonuclease NcoI; D015252:Deoxyribonucleases, Type II Site-Specific