Genetic Variations in Genomic HAS1 Direct Aberrant Pre-mRNA Splicing That Generates the Prognostically Significant HAS1Vb Intronic Splice Variant

Hyaluronan synthase 1 splice variants (HAS1Va, Vb and Vc) have been previously identified in multiple myeloma (MM) and Waldentsrom’s macroglobulinemia (WM) in addition to HAS1 full length (FL) transcript, and overexpression of HAS1Vb correlates with poor survival in MM (Adamia, et al, Blood2005; 105(12): 4836–44). Unlike HAS1FL that has 5 exons, the aberrantly spliced HAS1Vb transcript excludes exon 4, but retains the last 59 bp of intron 4. Sequence analysis of HAS1 intron 4 (2.1 kb) in MM and WM, but not in B-CLL, reveals frequent point mutations and other genetic variations in genomic HAS1 from bone marrow and blood (Adamia et al, submitted). Intronic mutations have been shown to regulate and modulate RNA splicing. Although it is likely that the mutations identified in HAS1 contribute to aberrant splicing events, an in vitro splicing assay was established to confirm that genomic HAS1 from patient DNA directed the aberrant splicing of HAS1 pre-mRNA to generate HAS1Vb. A 3.7 kb long HAS1 minigene extending from exon 3 to exon 5 was amplified from a WM sample in which HAS1Vb was expressed. Sequence comparison of this minigene to the germline indicated two point mutations in intron 4 (G >A at positions 13 and A>G at position 1837 downstream of exon 4), a T insertion within the T’s stretch, 1708 bp downstream of exon 4 and an insertion of 2 extra TTTA repeats, 1861 bp downstream of exon 4. There were no mutations identified in intron 3 of this patient. The minigene was fused to the upstream HAS1 cDNA sequence in a mammalian expression vector, pcDNA3, and transfected into HeLa cells, which do not otherwise express either HAS1 or HAS1 splice isoforms. RNA splicing as determined by RT-PCR indicated that both constitutive and aberrant splicing of HAS1, in particular, HAS1Vb, occurred in transfected cells, indicating a correct assembly of the construct and the presence in HeLa cells of the required spliceosome components. It also suggests that HAS1 minigene sequences used in this analysis provided cis-acting elements required for HAS1Vb production and that the aberrant HAS1 splicing is not restricted by B cell specific trans-acting elements. Site-directed mutagenesis of intron 4 is currently under investigation to determine the significance of each point mutation/insertion in the HAS1 clone studied. In combination with sequence information of HAS1 intron 4 compiled from sequencing data from a large number of MM and WM patients generated in this laboratory, future site-directed mutagenesis and deletion analysis will target on intron 4 subregions where mutations are shown to cluster. Overall, our studies will unveil the essential cis-acting elements that regulate the aberrant splicing of HAS1Vb. Our work to date confirms that intronic mutations in HAS1 direct aberrant HAS1 pre-mRNA splicing to exclude exon 4 and include a fragment of intron 4, thereby generating an intronic splice variant previously shown to correlate with poor survival in MM. This suggests that in MM and WM but not B-CLL, inherited and somatic variations in genomic HAS1 may contribute to early events in the oncogenic processes giving rise to these malignancies.