Germline and Somatic Mutations in the Hyaluronan Synthase–1 (HAS1) Gene May Contribute to Oncogenesis in Multiple Myeloma (MM) and Waldenstrom’s Macroglobulinemia (WM).
Authors: Adamia Sophia , Reichert Amanda , Ghosh Anirban , Hodges Jennifer , Pilarski Patrick , Treon Steven , Mant Michael J. , Reiman Tony , Belch Andrew R. , Pilarski Linda M. 
Published: April 2020

Abstract
In MM and WM, we identified aberrant HAS1 splice variants that were absent from normal donors (HD) and B-CLL. Here we sequenced multiple subclones from multiple cell subsets to show that aberrant HAS1 splicing results from cryptic splice sites activation. Aberrant splicing defects are the consequences of genetic variations (GVs) detected in the sequence of classical splicing elements as well as within exons and introns. To investigate HAS1 splicing in MM and WM patients, we sequenced the HAS1 gene segments involved in abnormal splicing events. HAS1 from buccal epithelial cells (BEC) represented the host genotype, and hematopoietic progenitors (HP), T, B and plasma cells (PC) as the normal and malignant components of the hematopoietic lineage in MM/WM. 197 GV were found in 16 MM or WM patients, but none in 9B-CLL, MGUS or HD. We found 60 germline (defined as present in BEC and hematopoietic cells) and 137 somatic GV (defined as GV found in HP, T, B and/or PC, but absent from BEC). These somatic GV include 97 tumor-specific GV found in MM and/or WM B and PC and 40 hematopoietic origin GV identified in HP, T, B and PC, but not in BEC. Some GV were recurrent, detected in more than one patient. Recurrent GV (24 in MM and 22 in WM) included both germline and somatic GVs, 6 tumor-specific, 6 hematopoietic and 14 germline origin GV, as well as 20 NCBI-SNPs. The distribution of GV indicated that some of the recurrent germline and somatic GV are restricted to MM, some are restricted to WM and some are shared by both MM and WM. None were found in B-CLL, MGUS or HD. The patterns of germline GV observed in MM and WM suggests that MM and WM patients, but not B-CLL, inherit recurrent germline GV that are necessary but not sufficient for progression to malignancy. Acquisition of recurrent, somatic HAS1 GV in HP further increases the risk of developing MM or WM. Transformation may become inevitable when tumor-specific recurrent GV are acquired. Interestingly, we detected increased homozygosity for the mutated allele of some germline GV in all cell types (PC, B, T, HPs and BECs) from MM and/or WM patients. These GV were detected in 75-90% of subclones analyzed. Mutational analysis of minigene constructs demonstrated a distribution of GV as clusters in the vicinity of HAS1splicing elements, allowing us to classify them as “splicing mutations”. This is supported by an in vitro splicing assay, which confirmed that a combination of germline and somatic GVs leads to aberrant HAS1 splicing (see abstract by Kriangkum et al., ASH 2007). Our study demonstrates that the impact of inherited and acquired GV on HAS1 gene splicing is manifested only in the context of accompanying tumor-specific HAS1 GV, that in combination give rise to the clinically significant aberrant splicing of HAS1. It also indicates that MM and WM are closely related at the genomic level, with the same recurrent somatic mutations independently arising in both diseases. Accumulation of somatically acquired HAS1 mutations in HP, in the context of inherited predispositions to MM and WM may represent a very early stage of pre-malignant development. Similar to leukemias, initiation events that contribute to MM and WM pathogenesis may arise from mutations which first accumulate during the non-malignant or pre-malignant stages of hematopoietic differentiation in progenitor cells.