•
Applications in Genetics
ered as mechanically and functionally distinct. In recent years, it has been realized that these phenomena have much in common and that Lymphotoxin-a, Lymphotoxin-13, TNF-a, and their multiple receptors participate in lymphoid organ
development and chronic inflammation [23].
TNF-a and TNF-13 bind to members of the same TNF receptor family, however, not on the same binding domains and thus cause partially comparable but also
partially different biological effects [27].
The TNF-a and TNF-f3 genes are located in tandem on human chromosome
6p21.3 within the class III region gene cluster of the major histocompatibility
complex {MHC),,,,250 kb centromeric of the HLA-B locus and 850 kb telomeric of
HLA-DR. Their high homology and close location in the genome, as well as evolutionary studies, suggest a common ancestor for both genes that duplicated during evolution [19].
Several polymorphisms have so far been noted in TNF-a and TNF-13 genes
{LT-a gene).1\vo promoter polymorphisms in the TNF-a gene are relatively frequent: G-238A at -238 relative to the transcriptional start site is found in about 5%,
G-308A at -308 relative to the transcriptional start site in about 30% of Caucasians,
and both modulate in vitro production of TNF-a by mononuclear cells (NCBI GenBank Accession #111698). The -308A allele of the promoter polymorphism is part
of the extended haplotype HLA-AI-B8-DR3-DQ2, which is associated with autoimmunity and high TNF production. The TNF-13 A329G polymorphism, identical to
the Nco I RFLP in intron 1 (NCB! GenBank Accession #M55913; NcoI will cut at
position 324), is even more common (40%-45%) [1]. Clinically, these polymorphisms have so far been associated with septic shock and survival of sepsis, various
autoimmune diseases (insulin-dependent diabetes mellitus type I [3] and lupus
erythematosus [21], Morbus Crohn, rheumatoid arthritis [2]), atopy [5] and asthma
[13], an increased risk of rejection after kidney transplantation and the response to
pathogens such as hepatitis Band C viruses [9,10], and chlamydia trachomatis.
In our study we examined correlations of these TNF polymorphisms with the
risk of developing sepsis after severe poly trauma. We found the -238A variant of
the TNF-a gene associated with sepsis development, but no correlation with the
G-308A polymorphism of the same gene and with A329G in the TNF-f3 gene [8],
using the LightCycler to detect these polymorphisms.
The methods used are described in detail in the following section.
Materials
Equipment LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
Oligo Primer Analysis Software (MedProbe, Oslo, Norway)
Reagents Amplification Primers and Hybridization Probes combined in ToolSets
(Genes-4U, Neftenbach, Switzerland, www.Genes-4U.com)
Qiagen QIAmp Blood kit (Hilden, Germany)
LightCycler-DNA Master Hybridization Probes (Roche Diagnostics, Mannheim,
Germany)
Applications in Genetics
ered as mechanically and functionally distinct. In recent years, it has been realized that these phenomena have much in common and that Lymphotoxin-a, Lymphotoxin-13, TNF-a, and their multiple receptors participate in lymphoid organ
development and chronic inflammation [23].
TNF-a and TNF-13 bind to members of the same TNF receptor family, however, not on the same binding domains and thus cause partially comparable but also
partially different biological effects [27].
The TNF-a and TNF-f3 genes are located in tandem on human chromosome
6p21.3 within the class III region gene cluster of the major histocompatibility
complex {MHC),,,,250 kb centromeric of the HLA-B locus and 850 kb telomeric of
HLA-DR. Their high homology and close location in the genome, as well as evolutionary studies, suggest a common ancestor for both genes that duplicated during evolution [19].
Several polymorphisms have so far been noted in TNF-a and TNF-13 genes
{LT-a gene).1\vo promoter polymorphisms in the TNF-a gene are relatively frequent: G-238A at -238 relative to the transcriptional start site is found in about 5%,
G-308A at -308 relative to the transcriptional start site in about 30% of Caucasians,
and both modulate in vitro production of TNF-a by mononuclear cells (NCBI GenBank Accession #111698). The -308A allele of the promoter polymorphism is part
of the extended haplotype HLA-AI-B8-DR3-DQ2, which is associated with autoimmunity and high TNF production. The TNF-13 A329G polymorphism, identical to
the Nco I RFLP in intron 1 (NCB! GenBank Accession #M55913; NcoI will cut at
position 324), is even more common (40%-45%) [1]. Clinically, these polymorphisms have so far been associated with septic shock and survival of sepsis, various
autoimmune diseases (insulin-dependent diabetes mellitus type I [3] and lupus
erythematosus [21], Morbus Crohn, rheumatoid arthritis [2]), atopy [5] and asthma
[13], an increased risk of rejection after kidney transplantation and the response to
pathogens such as hepatitis Band C viruses [9,10], and chlamydia trachomatis.
In our study we examined correlations of these TNF polymorphisms with the
risk of developing sepsis after severe poly trauma. We found the -238A variant of
the TNF-a gene associated with sepsis development, but no correlation with the
G-308A polymorphism of the same gene and with A329G in the TNF-f3 gene [8],
using the LightCycler to detect these polymorphisms.
The methods used are described in detail in the following section.
Materials
Equipment LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
Oligo Primer Analysis Software (MedProbe, Oslo, Norway)
Reagents Amplification Primers and Hybridization Probes combined in ToolSets
(Genes-4U, Neftenbach, Switzerland, www.Genes-4U.com)
Qiagen QIAmp Blood kit (Hilden, Germany)
LightCycler-DNA Master Hybridization Probes (Roche Diagnostics, Mannheim,
Germany)