Single Nucleotide Polymorphisms (SNPs) and Recurrent Mutations in the Hyaluronan Synthase 1 (HAS1) Gene May Predispose to Multiple Myeloma (MM) and Waldenstrom’s Macroglobulinemia (WM).
Authors: Adamia Sophia , Treon Steve , Santos Daniel , Hunter Zachary , Mant Michael J. , Reiman Tony , Belch Andrew R. , Pilarski Linda M. 
Published: April 2020

Abstract
The hyaluronan synthase 1 (HAS1) gene, which maps to chromosomal location 19 q3.14 undergoes aberrant intronic splicing in malignant B cells from MM and WM patients. We previously detected up-regulation of HAS1 gene in MM and WM, and identified three aberrantly spliced transcripts of this gene, termed HAS1Va, HAS1Vb and HAS1Vc. With a much larger patient cohort, statistical analysis of samples taken from 201 untreated MM patients showed that expression of HAS1Vb strongly correlates with poor survival (P=0.005). Furthermore, for MM patients with lytic bone disease, those patients having both lytic lesions and expression of HAS1Vb had statistically shorter survival than those with lytic lesions but lacking HAS1Vb (p=0.02). Our gene expression analysis at the single cell level demonstrates that 76–97% of individual CD20+ IgM+ WM cells obtained from BM aspirates and PBMC expressed HAS1Va or HAS1Vb variants. Exon skipping and partial intron retention in the HAS1 gene, detected in WM and MM cells, can result from activation of cryptic splicing elements. In turn, activation of cryptic splicing elements can be promoted by mutations, including SNPs, which serve as frequent genetic markers along the chromosome. We analyzed the frequency of the HAS1 833 A/G SNP located upstream of alternatively spliced exon 4, on exon 3. Bioinformatic analysis demonstrated that HAS1 833 A/G SNP overlaps with an exonic splicing enhancer motif of HAS1 and in the mutated allele this exonic splicing enhancer is abrogated. The genotype of G/G and A/G was found respectively in 90% and 10% of 60 WM cases, 85% and 15% of 117 MM cases and 40% and 60% of 90 cancer-free cases. Our results demonstrate that the genotype G/G is significantly more frequent in WM and MM patients than in cancer free controls (p=0.0001for WM and p= 0.00003 MM). The genotype A/A was not detected in any of the 267 individuals tested to date, and is presumed lethal. Further, in MM and WM B cells, we identified recurrent mutations on HAS1 intron 4 that include alterations of a key splicing element, polypyrimidine tract that creates and/or activates a new splice site in precisely the position required for the splicing events that create HAS1Vb. Bioinformatic analysis indicates that these recurrent genomic variations in intron 4 alter splisosome assembly, leading to aberrant splicing of HAS1 and the predicted impact on malignant disease and patient survival. We speculate that SNPs in HAS1 provide the predisposing elements for MM and WM, and additional mutations within the genome of emerging malignant B cells are required for progression to malignant disease. Thus, aberrant splicing of the HAS1 gene in MM and MW patients may results from the presence of polymorphisms and recurrent mutations in the HAS1 gene. Our genotyping analysis suggests that SNPs and mutations in the HAS1 gene may contribute to oncogenic events in WM or MM.