Introduction: A genome-wide scan of linkage with venous thrombosis in a thrombophilic kindred with type I protein C deficiency identified a candidate region on chromosome 11q23 (P<0.0001). Located at the linkage peak is PAFAH1B2, the β-subunit of the intracellular form of PAFAH. Intracellular PAFAH is a heterodimer of alpha and beta subunits both of which have catalytic activity. PAFAH reduces intracellular PAF levels and a reduction in active PAFAH likely leads to an enhanced inflammatory response at the vascular wall. Resequencing experiments revealed a novel splice variant for the intracellular form of PAFAH. The current study characterizes this novel splice variant that results in the deletion of exon 6 and its catalytic domain and the introduction of an alternative 7th exon.
Methods: Identification of novel mRNA splice variants was achieved by sequencing PCR products obtained from amplifying known regions of PAFAH 1B2 mRNA. Confirmation of the exon 5 to 7 splice and exon 7 polyadenylation were obtained from sequencing 3′RACE products. Protein extracts were resolved by SDS- PAGE and Western blotted.
Results: An alternative splice form of PAFAH 1B2 was isolated and sequenced. This isoform was observed in cDNA from human liver, moncytes, and T and B lymphocytes using PCR. This variant contains the first five exons of the native form protein and replaces exon 6 with an alternative 7th exon located downstream of exon 6. Exon 6 contains two residues involved in the protein’s active site triad, Asp and His, and are absent in exon 7. Computer modeling suggests replacement of exon 6 by exon 7 would abolish the active site and possibly alter the proteins’ ability to dimerize. Preliminary Western blots from selected tissues demonstrated the presence in liver of a new band consistent with the predicted size of the variant.
Conclusions: We hypothesize that this alternative splice likely decreases the proteins ability to function as a regulator of inflammation. We are presently carrying out gene expression analysis to determine the relative levels of the splice variant in different tissues and changes in expression associated with activation of leukocytes.