Functional and Familial Risk of B Lineage Malignancies in Patients Carrying Single Nucleotide Polymorphisms (SNPs) in the Hyaluronan Synthase 1 Gene (HAS1),

Abstract 3930

Risk associations with genetic changes, especially SNPs, can be most meaningfully interpreted when the functional relevance of the involved gene(s) is known. Further, using targeted sequencing to detect SNPs provides for unequivocal allele “calling” in each patient, as well as identification of any linked low penetrance mutations that might influence risk. HAS1 is overexpressed and aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulimenia (WM), but not in healthy donors (HD); HAS1Vb correlated with reduced survival in a cohort of MM patients¹. In transfectants, HAS1 variants are oncogenic in vivo and/or in vitro². As shown here, a set of three intron 3 SNPs in linkage disequilbrium have significantly different genotype and allele frequencies and robust hazard ratios in people with B lineage malignancies as compared to age matched controls and to a set of unlinked exon 3 HAS1 SNPs. In all patient and control groups, all five SNPs met Hardy-Weinberg equilibrium. We sequenced an 850bp segment of the HAS1 gene (exon 3, intron 3) from PBMC of 307 Caucasian individuals, including 86 MM, 70 monoclonal gammopathy of undetermined significance (MGUS), 25 WM, 40 B chronic lymphocytic leukemia (CLL), 15 affected and 21 unaffected members of a monoclonal gammopathy prone Icelandic kindred, and 60 age-matched HD. Using direct or subclone sequencing, we evaluated the frequencies of NCBI designated minor alleles for the 5 SNPs. Bioinformatics and in vitro mutagenesis experiments confirmed that intron 3 plays a central role in clinically relevant splice site selection and aberrant intronic HAS1 splicing³. The linked intron 3 HAS1 SNPs (rs11084110, rs11084109 and rs11669079) in patient groups have significantly different host genotype and allele frequencies from those of HD. The frequencies of the unlinked exon 3 HAS1 polymorphisms (rs61736495, rs11084111) on the same 850bp amplicon were not significantly different between patients and HD, providing internal controls for sequencing artefacts. Associations between risk of B cell malignancy and HAS1 SNPs were evaluated using the logistic regression model. Compared to age matched controls, the HAS1 intron 3 SNPs were significantly associated with MM and MGUS for genotype frequencies (p.01 to.05) and allele frequencies (p.01 to.0007); the association was even stronger for CLL and WM (genotype frequencies p<-10E5, allele frequencies p<-10E5). Exon 3 HAS1 polymorphisms were not significantly different in patients as compared to HD. To evaluate individuals with common ancestry, we sequenced the same HAS1 region from members of a monoclonal gammopathy prone Icelandic kindred. In this kindred, the frequencies of intron 3 SNPs of affected members (clinical cases and those with functional B cell abnormality) were significantly different from those of unaffected members (genotype frequencies p=0.01, allele frequencies p=.001); thus genotype and allele frequencies for the intron 3 HAS1 SNPs remain statistically significant even in this small but relatively homogeneous cohort. In an ODDS-ratio analysis of pooled data from all patient and control groups, OR_trend ranged from 4.7 to 6.8 (p=0.0001). These robust hazard ratios together with the familial analysis exclude the influence of cryptic population stratification. Risk predictions may be directly correlated with the identified SNPs, or alternatively may result from unknown mutation(s) in linkage disequilibrium with the detected SNPs. To resolve these possibilities, highly sensitive targeted sequencing provides a degree of certainty not possible with genome wide association studies. The risk associations observed here are directly correlated with the indicated SNPs because no other significant variations were identified by sequencing. The functional relevance of the associations reported here is supported by in vitro mutagenesis of intron 3, which shows a profound impact of SNPs/mutations on alternative splicing and splice site selection that leads to aberrant intronic splicing of the type seen in patients. Although multiple genes are certain to be involved, this work supports the idea that the minor alleles for the HAS1 intron 3 SNPs have a strong functional impact on epigenetic events, particularly aberrant pre-mRNA splicing, that contribute to the malignant phenotype in B lineage cancers.

1. Blood 2005 105:4836 2. JBC 2009 285:18840 3. Blood 2008 512:5111