Identification of the nucleotide sequence of the lipoprotein lipase gene as well as its role in the development of hyperlipidemia and pancreatitis in the Miniature Schnauzer

摘要

Lipoprotein Lipase (LPL) is a key enzyme in lipid transport. It catalyses the hydrolysis of thetriacylglycerol component of chylomicrons and very low-density lipoproteins (VLDL), providingnon-esterified fatty acids for tissue utilization. The gene encoding for LPL has already beenidentified in several species except the dog. Mutations of the human LPL-gene have been shown tocause partial or complete malfunction of the enzyme, resulting in accumulation of lipoproteins in theblood. This condition is called familial LPL deficiency. LPL malfunction results inhyperlipoproteinemia, recurrent acute pancreatitis, and ultimately pancreatic insufficiency.Several authors have postulated a genetic cause for pancreatitis in the Miniature Schnauzer. Anidiopathic increase in serum triglyceride concentration can also be found in this breed.Based on these findings we were evaluating a possible role of the lipoprotein lipase gene in thedevelopment of pancreatitis and hyperlipidemia in the Miniature Schnauzer. First, we identified thegenetic sequence of the LPL gene in the dog. We determined clones on the Trace Archive databasefor the canine genome project that contain the genomic sequence of a particular exon as well as itsadjacent intronic regions. Based on these findings we designed primers for each exon using thesoftware Netprimer (www.premierbiosoft.com/netprimer/index.html). Canine subjects were chosenfrom a pool of 170 Miniature Schnauzers from the database at the Gastrointestinal Laboratory atTexas A&M University. Based on clinical history, serum cPLI concentrations, and serum triglycerideconcentrations 21 Miniature Schnauzers were chosen and were selected into a clinically normalcontrol group (9 dogs) and an affected group (12 dogs). DNA was then collected from either whiteblood cells or mucosal cells of these dogs. After PCR optimization, exon 1 through 9 including theadjacent intronic regions were amplified in all dogs using MasterAmp Extra – Long PCR Kit(Epicentre, WI, USA) and were sequenced in triplicates. Differences in the nucleotide sequenceswere then compared among the two groups. 10 exonic SNPs and 9 intronic SNPs were identified.Upon analysis, none of these variations could be associated with the disease status.We conclude that pancreatitis associated with hyperlipidemia in the Miniature Schnauzer is notlinked to mutations of the lipoprotein lipase gene or its splicing regions.