Genetics of PLCG2 expression and splicing relative to Alzheimer’s disease risk

Background PLCG2 is associated with the risk of Alzheimer’s disease (AD) through a rare missense polymorphism, rs72824905 (P522R) as well as a common variant, rs12445675, within a long non-coding RNA adjacent to PLCG2. Elucidating the impact of genetics on PLCG2 expression and splicing will provide insights into the role of PLCG2 in AD risk and, potentially, treatments that might reduce AD risk. Objective To evaluate PLCG2 expression and splicing as a function of AD genetics. Methods PLCG2 isoform expression was detected by PCR and quantified by qPCR in AD and non-AD brain samples and in blood buffy coat samples. The function of a genetic variant, rs107164, was tested by using a minigene approach with both alleles in murine BV-2 microglial cells. The impact of ectopic splicing factor expression on PLCG2 minigene splicing was also tested in BV-2 cells. The extent that endogenous levels of a novel PLCG2 mRNA isoform lacking 65 bp within exon 28 (D65-PLCG2) were affected by nonsense mediated decay (NMD) was determined by using cycloheximide in vitro. Lastly, whether D65-PLCG2 manifested a Ca + 2 response similar to PLCG2 was tested by comparing D65-PLCG2-GFP and PLCG2-GFP fusion proteins in transfected HEK293 cells. Results We report PLCG2 isoforms that include (i) a transcript that replaces PLCG2 exon 1 with sequence from an adjacent long noncoding (LNC) RNA (LNC-PLCG2) and (ii) a transcript that lacks 65 bp from the beginning of exon 28 (D65-PLCG2). The ratio of LNC-PLCG2 to canonical PLCG2 was associated with rs12445675 genotype in both human brain and buffy coat samples. The proportion of PLCG2 expressed as D65-PLCG2 was increased by the T allele of rs1071644, a T/C SNP within the 65bp variably spliced portion of exon 28. This SNP was demonstrated to be functional in a minigene splicing assay. Moreover, the rs1071644-T allele was found to be associated with increased AD risk, independent of rs72824905 (P522R) and rs12445675. D65-PLCG2 was susceptible to nonsense mediated RNA decay. D65-PLCG2 was not responsive to Ca^{+ 2} in a fashion similar to that observed for PLCG2. Hence, the rs1071644-T allele appears to increase AD risk by increasing the proportion of PLCG2 expressed as D65-PLCG2, representing a loss of PLCG2 function. Conclusions We report that two AD genetic risk factors, rs12445675 and rs1071644, affect AD risk by impacting the LNC-PLCG2 to PLCG2 ratio and PLCG2 exon 28 splicing, respectively.