The variant rs77559646 associated with aggressive prostate cancer disrupts ANO7 mRNA splicing and protein expression

Abstract

Prostate cancer is among the most common cancers in men, with a large fraction of the individual risk attributable to heritable factors. A majority of the diagnosed cases does not lead to a lethal disease, and hence biological markers that can distinguish between indolent and fatal forms of the disease are of great importance for guiding treatment decisions. Although over 300 genetic variants are known to be associated with prostate cancer risk, few have been associated with the risk of an aggressive disease. One such variant is rs77559646 located in ANO7. This variant has a dual function. It constitutes a missense mutation in the short isoform of ANO7 and a splice region mutation in full-length ANO7. In this study, we have analyzed the impact of the variant allele of rs77559646 on ANO7 mRNA splicing using a minigene splicing assay and by performing splicing analysis with the tools IRFinder, rMATS and LeafCutter on RNA-Seq data from prostate tissue of six rs77559646 variant allele carriers and 43 non-carriers. The results revealed a severe disruption of ANO7 mRNA splicing in rs77559646 variant allele carriers. Immunohistochemical analysis of prostate samples from patients homozygous for the rs77559646 variant allele demonstrated a loss of apically localized ANO7 protein. Our study is the first to provide a mechanistic explanation for the impact of a prostate cancer risk SNP on ANO7 protein production. Furthermore, the rs77559646 variant is the first known germline loss-of-function mutation described for ANO7. We suggest that loss of ANO7 contributes to prostate cancer progression.

Transcripts of the Prostate Cancer-Associated Gene ANO7 Are Retained in the Nuclei of Prostatic Epithelial Cells

“…Our observations suggest that in the normal prostatic epithelium, ANO7 is expressed in luminal cells but not in basal cells, as the RNA-FISH signal was not detected in the basal layer of the epithelium ( Figure 2 D). Anti-ANO7 staining in both basal and luminal cells has been reported [ 19 , 28 ]. The antibody used in both of these studies, however, is not completely specific to ANO7 ( (accessed on 7 December 2022)), and the signal in basal cells was also observed in patients homozygous for a mutation that causes a severe splicing defect in ANO7 [ 28 ].…”

“…Anti-ANO7 staining in both basal and luminal cells has been reported [ 19 , 28 ]. The antibody used in both of these studies, however, is not completely specific to ANO7 ( (accessed on 7 December 2022)), and the signal in basal cells was also observed in patients homozygous for a mutation that causes a severe splicing defect in ANO7 [ 28 ]. Bera et al [ 7 ] concluded from in situ hybridization data that ANO7 mRNA is expressed in basal and terminal epithelial cells of the epithelium.…”

“…We have recently shown that the ANO7 PrCa risk SNP rs77559646, which is located in the intron 4 splice donor region, causes a severe splicing defect. Nevertheless, equal levels of ANO7 mRNA appeared to be expressed from both copies of the gene in prostate tissue [ 28 ]. We therefore hypothesized that a large fraction of ANO7 mRNA might be nuclearly retained without being degraded, given that an aberrantly spliced RNA with in-frame premature termination codons that has been exported to the cytoplasm should be targeted for degradation [ 31 ].…”

“…Intron retention is a known mechanism behind the nuclear accumulation of transcripts [ 37 , 38 , 39 ]. We have shown that some introns in ANO7 are partially retained in patient RNA-Seq data [ 28 ]. Thus, we hypothesize that the retained introns in ANO7 may harbor nuclear retention elements.…”

“…The immunohistochemistry for the TMA sections was performed as described [ 28 ]. The primary antibody against ANO7 was HPA035730 (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:200.…”