Two high-risk susceptibility loci at 6p25.3 and 14q32.13 for Waldenström macroglobulinemia

Abstract

Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) is a rare, chronic B-cell lymphoma with high heritability. We conduct a two-stage genome-wide association study of WM/LPL in 530 unrelated cases and 4362 controls of European ancestry and identify two high-risk loci associated with WM/LPL at 6p25.3 (rs116446171, near EXOC2 and IRF4; OR = 21.14, 95% CI: 14.40-31.03, P = 1.36 × 10^-54) and 14q32.13 (rs117410836, near TCL1; OR = 4.90, 95% CI: 3.45-6.96, P = 8.75 × 10^-19). Both risk alleles are observed at a low frequency among controls (~2-3%) and occur in excess in affected cases within families. In silico data suggest that rs116446171 may have functional importance, and in functional studies, we demonstrate increased reporter transcription and proliferation in cells transduced with the 6p25.3 risk allele. Although further studies are needed to fully elucidate underlying biological mechanisms, together these loci explain 4% of the familial risk and provide insights into genetic susceptibility to this malignancy.

Fig. 1

Regional association plots of two SNPs associated with the risk of WM/LPL. a Chromosome 6p25.3 (rs116446171) and b chromosome 14q32.13 (rs117410836). Shown are the −log₁₀ association P-values from the discovery log-additive genetic model for all SNPs in the region (dots) and combined discovery and replication fixed-effects analysis (diamonds). The lead SNPs are shown in purple, with results from both the discovery (small diamonds) and combined (large diamonds) analyses. Estimated recombination rates (from 1000 Genomes) are plotted in blue. The SNPs surrounding the most significant SNP are color-coded to reflect their correlation with this SNP. Pairwise r² values are from 1000 Genomes European data. Locations of recombination hotspots are depicted by peaks corresponding to the rate of recombination. Genes, position of exons and direction of exons and direction of transcription from UCSC genome browser (http://genome.ucsc.edu) are denoted. Plots were generated using LocusZoom (http://csg.sph.umich.edu/locuszoom)

Fig. 2

Genomic position and alignments of rs116446171 to miRs. a Schematic representation of the position of rs116446171 relative to the 3′UTR of EXOC2 on chromosome 6 and b alignments of rs116446171 wild type and risk variants with the binding sites of microRNAs, miR-378a-5p and miR-324-3p

Fig. 3

The rs116446171 variants affect reporter activity and cell proliferation. a EGFP reporter activity in HEK293T stably transduced cell lines. Cells transduced with the risk variant (G) showed significantly increased fluorescence levels of EGFP compared to the cell lines transduced with the wild type (WT, (C); P = 0.012). Cells transduced with the Null (Δ) had decreased EGFP fluorescence (P = 0.054, n = 14), and cells transduced with the commercial 3′UTR of EXOC2 showed significantly decreased EGFP fluorescence (P < 0.0001, n = 14). Data are expressed as mean fold change relative to the cells transduced with the vector, ±standard error of the mean (s.e.m.), n = 14 replicates. **P < 0.01, ****P < 0.0001. b Quantitative PCR analysis of EGFP transcripts in HEK293T stably transduced cell lines. Significant changes of EGFP mRNA levels were detected in cells harboring the variant allele compared to the cells harboring the wild-type allele (P = 0.031). Cells harboring the Null allele had reduced EGFP transcripts levels (P = 0.036). Data are expressed as mean % change relative to the endogenous controls, ±s.e.m., n = 9 replicates for each experiment. *P < 0.05. c Proliferation assay of cells harboring rs116446171, the deletion (Null) of an 18-bp segment centered on rs116446171, and the commercial 3′UTR reporter. The cell line transduced with the variant allele showed significantly increased cell proliferation compared to the cell lines transduced with the EXOC2 3′UTR, the WT and the Null. Data are expressed as mean fold change of the cell line in the day seeded, ±s.e.m., n = 9 replicates. ****P < 0.0001. All P-values were calculated with unpaired t-test

Fig. 4

EGFP reporter assay of interactions with microRNAs. Transient transfections with either the PremiR-378a-5p or PremiR-324-3p expression plasmid reduced the EGFP protein expression in cells harboring the wild type or variant allele equally (i.e., resulted in similar fold reductions). Transfection of PremiR-324-3p significantly increased the EGFP fluorescence in the cells harboring the variant allele compared to the wild type (P = 0.040). Data are expressed as mean fold change relative to the cells transfected with the pLV-miR vector, ±s.e.m., n = 9 replicates. P-values were calculated with unpaired t-test. *P < 0.05. N.B. the scale of the fold change on the Y axis is <1.0

Fig. 5

Dose-dependent effect of rs116446171 variants on reporter transcription and cell proliferation. a Quantitative PCR analysis of EGFP transcripts in stably transduced cells with tandem repeats of the rs116446171 variant allele. The variant allele was inserted within the EXOC2 3′UTR region as a single copy or as two, four and eight repeats in either cis or trans orientation. Data are expressed as mean fold change of the endogenous controls, ±s.e.m., n = 9 replicates. ****P < 0.0001. b Proliferation assay of cells transduced with tandem repeats of the variant allele. Cells harboring eight tandem repeats proliferate significantly faster than cells harboring four tandem repeats, and cells harboring four tandem repeats proliferate significantly faster than cells harboring two or one repeat. Data are expressed as mean fold change of the cell line in the day seeded, ±s.e.m., n = 9 replicates. ****P < 0.0001. All P-values were calculated with unpaired t-test