Two bi-allelic single nucleotide polymorphisms within the promoter region of the horse tumour necrosis factor alpha gene.

The research article discusses the identification of two single nucleotide changes, or polymorphisms, in the promoter region of the horse tumour necrosis factor alpha gene, a region that is key to gene expression.

Identification of Polymorphisms in Horse TNF-Alpha Promoter

• Researchers designed special “primers” – short stretches of DNA used to initiate a process called polymerase chain reaction (PCR) that amplifies specific DNA sequences – based on known sequences within the 5′ untranslated region (UTR) of the human and horse tumour necrosis factor alpha (TNF-alpha) genes.
• Using these primers, a 522-base pair product was amplified and sequenced from five independent PCRs obtained from three different horses of three different breeds.
• The sequences obtained matched closely with the TNF-alpha promoter regions of other species and expanded our knowledge of the horse TNF-alpha sequence 230 nucleotides further than previous entries in GenBank, a genetic sequence database.

Development of a New Primer and Discovery of Allelic Variants

• Based on the sequence data obtained, the scientists designed a new horse-specific forward primer that amplified a smaller, 213 base-pair PCR product.
• They screened this product for genetic variation using a technique known as single-strand conformation polymorphism (SSCP), which involves detecting differences in the physical shape of DNA strands caused by changes in their sequence.
• This screening led to the discovery of three allelic variants of the horse TNF-alpha gene, differing in sequence at particular points. These sequences were submitted to GenBank with the accession numbers ADF 349558-60.
• The differences between the three variants were traced back to two single nucleotide polymorphisms (SNPs) – specific points in a DNA sequence where variants have a different DNA base. These polymorphisms were found at positions 137 and 147.

Analysis of Breed-Specific Allelic Frequencies

• The researchers also noticed that the frequency of the different allelic variants differs between horse breeds.
• In particular, the Old Kladruber and Akhal-Teke breeds showed notable differences in the distribution of the variants.

This work provides a better understanding of the genetic makeup of the horse’s TNF-alpha gene and provides a foundation for future studies on the potential role of these polymorphisms in horse health and disease.