Association of Common Variants of TNFSF13 and TNFRSF13B Genes with CLL Risk and Clinical Picture, as Well as Expression of Their Products—APRIL and TACI Molecules

Simple Summary A growing body of evidence reported in literature suggests an important role of APRIL in the development and pathogenesis of chronic lymphocytic leukaemia (CLL), in particular elevated levels of soluble APRIL (sAPRIL) and overexpression of its mRNA have been noticed in CLL cells. Moreover, TACI expression has been found to improve the survival ability of CLL cells protecting them from apoptosis in vitro. On the other hand, there are evidence supporting the existence of a genetic predisposition to develop CLL. These data prompted us to investigate the influence of genetic variants of TNFSF13 and TNFRSF13B on the expression level of proteins encoded by these genes and to investigate the association between these variants an CLL risk. Abstract Interactions between APRIL (TNFSF13) and its receptor TACI (TNFRSF13B) are implicated in providing survival benefits for chronic lymphocytic leukaemia (CLL) cells. Here we explored the relationship between TNFSF13 and TNFRSF13B SNPs and expression of APRIL and TACI molecules and performed extended case-control study to evaluate earlier observations. Expression of APRIL and TACI was detected by FACS for 72 and 145 patients, respectively, and soluble APRIL was measured by ELISA in plasma of 122 patients. Genotypes were determined in 439 CLL patients and 477 control subjects with TaqMan Assays or restriction fragment length polymorphism (RFLP). The rs4968210GG genotype of TNFSF13 was associated with a lower percentage of CD19+APRIL+ cells in CLL patients when compared to (AA + GA) genotypes (p-value = 0.027). Homozygosity at rs11078355 TNFRSF13B was associated with higher CD19+ TACI+ cell percentage in CLL patients (p-value = 0.036). The analysis of extended groups of patients and healthy controls confirmed the association of TNFSF13 rs3803800AA genotype with a higher CLL risk (OR = 2.13; CI95% = 1.21; 3.75; p-value = 0.007), while the possession of TNFRSF13B rs4985726G allele (CG + GG) genotype was associated with lower risk of CLL (OR = 0.69; CI95% = 0.51; 0.95; p-value = 0.02). Genetic variants of TNFSF13 and TNFRSF13B may have an impact on APRIL and TACI expression and may be considered as possible CLL risk factors.


Supplementary
Since elucidating the function of risk variants and variants associated with phenotypic features is an important step towards a better understanding of the biological processes involved in disease development and outcomes, we used publicly available sources, described below to examine a potential functional relevance of rs3803800G>A, rs4968210G>A, rs4985726C>G, and rs11078355A>G genetic variants.
Of note, TNFSF13 is located in close proximity to the TNFSF12 gene for another member of the TNFSF, namely the TNF-like weak inducer of apoptosis (TWEAK, TNFSF12). The adjacent localisation and the same transcriptional direction of these two genes enables the production of mRNA molecule (TNFSF12-TNFSF13) and protein (TWE-PRIL) consisted of the cytoplasmic/transmembrane and stalk domains of TWEAK and receptor binding domains of APRIL due to and intergenic splicing event.
Given the data provided by Bojarska-Junak et al. [3] showed a higher expression of APRIL mRNA and intracellular APRIL in peripheral blood (PB ) CD19 + leukemic cells than in PB CD19 + cells isolated from the control group, we checked if rs3803800 localizes to any regulatory region by applying the ENCODE dataset [4][5]. One matching candidate is the Cis Regulatory Element (cCRE) accession number EH38E1844485 (hg38), which was shown for rs3803800G>A with a proximal enhancer like signature (Supplementary Figure S2B,C, respectively). This cCRE was associated with TNFSF13 expression and according to ENCODE the IKZF1 transcription factor may bind within this cCRE, which was observed for GM12878 cell line. These data suggest that rs3803800 may potentially localize to regulatory region. The Supplementary Figure S2B,C presents rs3803800 surrounded by regulatory elements and TF binding sites (hg38). We have also performed an additional examination with application of GTEx [6] multi-tissue eQTLs analysis to see if this variant affect expression in other cells and tissues (Supplementary Figure S2D). The rs3803800 appeared to be significantly associated with TNFSF13 expression in many tissues (Meta-Analysis RE2: p-value=5.2x10 -55 ). This analysis showed that the minor allele A of rs3803800 (reference allele in GTEx) is in the majority of tissues associated with higher TNFSF13 expression.
rs4968210G>A variant constitutes G to A substitution in intron 5 of TNFSF12-TNFSF13 transcript as well as of TNFSF12 transcript. As described earlier, we observed that A allele is associated with a higher average percentage of CD19 + APRIL + CLL cells. Taking this into consideration we employed ENCODE [4][5] Figure S3a) [1][2] localize to regulatory elements. According to ENCODE, rs4968210 variant does not overlap any cCRE, but is located 981 bp from cCRE EH38E1844474 (hg38) which is predicted to have a distal enhancer like signature inter alia in B cells and GM12878 (Supplementary Figure S3B and 3C) cell line and is associated with TNFSF12-TNFSF13 mRNA expression. Additionally, another SNP in LD with rs4968210 (Supplementary Figure S3A) [1][2] namely rs12942590 is located within 221 bp distance of this cCRE (Supplementary Figure S3B and C). Accordingly, the transcription factors important for B cell biology such as EBF1, PAX5, SPI1, IKZF1, IKZF2, and BHLHE40 were shown to bind in this region (Supplementary Figure S3B and 3C).

to check if this variant or any variant in LD with it (Supplementary
The ENCODE data suggest that the impact of rs4968210 observed by us on phenotype of CD19 + B cells in relation to APRIL may be associated with the fact that rs4968210 and rs12942590 are located near potential regulatory element associated with B cells biology and TNFSF12-TNFSF13 expression. To further evaluate the association between rs4968210 and TNFSF12-TNFSF13 expression we performed an additional analysis with application of GTEx multi-tissue eQTLs analysis to see if this variant affect expression in other cells and tissues (Supplementary Figure S3D). The rs4968210 turned out to be significantly associated with TNFSF12 expression in many types of tissues (Meta -Analysis RE2: p-value=1.2x10 -94 ). This analysis indicates that the rs4968210G allele (reference allele in GTEx) is associated with higher TNFSF12 (TWEAK) expression. in the majority of tissues for that the data were available. However, in EBV-transformed lymphocytes an opposite effect can be observed, unfortunately this association was not significant. This is in line with our observation that patients with rs4968210GA and rs4968210AA genotypes had higher average percentage of CD19 + APRIL + CLL cells. The rs4968210 was also associated with TNFSF13 eQTL expression in multiple tissues (Meta -Analysis RE2: p-value=1.9x10 -11 ). The result of this analysis suggests that rs4968210 may be associated with allele specific expression which seems to be tissue specific (Supplementary Figure S3E).
The Supplementary Figure S3A presents SNPs in LD with rs4968210 and Figure S3B,C present 4968210 and rs12942590 surrounded by regulatory elements and TF binding sites (hg38) in PB B cells as well as in GM12878 cell line, respectively. rs4985726C>G variant is located in intron 1 of TNFRSF13B gene. According to HaploReg tool (v4.1) [1][2] this variant exists in LD with 10 intronic SNPs of TNFRSF13B and one missense rs34562254G>A (Pro251Leu) variant of TNFRSF13B (Supplementary Figure S4A). The rs34562254G>A (Pro251Leu) variant was predicted to be a possibly damaging variant by PolyPhen2 (Exome Variant server: 0.728; Ensembl: 0.476). Rs4985726C>G did not overlap with any cCRE both in PB B cells as well as in GM12878 cell line (Supplementary Figure S4B and S4C, respectively). Accordingly, we did not observe association between this variant and TACI expression in PB CLL cells. Similarly, analysis with GTEx portal [6] did not reveal significant eQTLs associated with rs4985726C>G. However, ENCODE [4][5] analysis for SNPs in LD with rs4985726 revealed data which may provide some potential explanation for the association of rs4985726 with the susceptibility to CLL risk observed by us. One of such SNPs, namely, rs57382045 did not overlap with any cCRE, however it is located 299 bp from EH38E1849614 cCRE (Supplementary Figure S4D,E). This cCRE was predicted to have distal enhancer-like signature in B cells with many TFs shown to bind in this region among others IKZF1 and IKZF2 (Supplementary Figure S4D). What is interesting, EH38E1849614 cCRE seems to be inactive (low DNase) in GM12878 cell line (Supplementary Figure  S4E). The Supplementary Figure 4A Figure S6A). Analysis with application of Human Splicing Finder (HSF) [7] showed that this variant may potentially cause alternation of splicing, by affecting the binding site for 9G8 (also known as SRSF7) splicing factor (Supplementary Figure S5A). Additionally, our studies revealed an association between genotypes of rs11078355A>G and expression of TACI receptor on CD19 + TACI + leukemic cells. Given that, we checked if rs11078355 and/or any variant in LD with it localize to any cCREs with possible impact on B cell biology. We did not find such cCREs (Supplementary Figure S6B,C). Application of GTEx portal [6] did not reveal significant eQTLs and sQTLs for rs11078355A>G