Turner Andrew K, Dotson Kennedy, Qiao Qi, Cain Kailey, Simpson James F, Fardo David W, Estus Steven
University of Kentucky.
Res Sq. 2025 Jun 19:rs.3.rs-6735123. doi: 10.21203/rs.3.rs-6735123/v1.
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 . 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.
To evaluate expression and splicing as a function of AD genetics.
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 mRNA isoform lacking 65 bp within exon 28 (D65-PLCG2) were affected by nonsense mediated decay (NMD) was determined by using cycloheximide . Lastly, whether manifested a Ca + 2 response similar to was tested by comparing D65-PLCG2-GFP and PLCG2-GFP fusion proteins in transfected HEK293 cells.
We report isoforms that include (i) a transcript that replaces exon 1 with sequence from an adjacent long noncoding (LNC) RNA () and (ii) a transcript that lacks 65 bp from the beginning of exon 28 (). The ratio of to canonical was associated with rs12445675 genotype in both human brain and buffy coat samples. The proportion of expressed as 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. was susceptible to nonsense mediated RNA decay. D65-PLCG2 was not responsive to Ca in a fashion similar to that observed for PLCG2. Hence, the rs1071644-T allele appears to increase AD risk by increasing the proportion of expressed as , representing a loss of PLCG2 function.
We report that two AD genetic risk factors, rs12445675 and rs1071644, affect AD risk by impacting the to ratio and exon 28 splicing, respectively.
磷脂酶Cγ2（PLCG2）通过一种罕见的错义多态性rs72824905（P522R）以及位于相邻长链非编码RNA内的一个常见变体rs12445675与阿尔茨海默病（AD）风险相关。阐明遗传学对PLCG2表达和剪接的影响将有助于深入了解PLCG2在AD风险中的作用，并可能为降低AD风险的治疗方法提供思路。
评估作为AD遗传学函数的PLCG2表达和剪接情况。
通过聚合酶链反应（PCR）检测AD和非AD脑样本以及血白细胞层样本中PLCG2亚型的表达，并通过定量PCR进行定量。采用小基因方法在小鼠BV-2小胶质细胞中测试遗传变体rs107164的功能，该方法使用了两个等位基因。还在BV-2细胞中测试了异位剪接因子表达对PLCG2小基因剪接的影响。通过使用环己酰亚胺确定缺乏外显子28内65 bp的新型PLCG2 mRNA亚型（D65-PLCG2）的内源性水平受无义介导衰变（NMD）影响的程度。最后，通过比较转染的人胚肾293（HEK293）细胞中的D65-PLCG2-绿色荧光蛋白（GFP）和PLCG2-GFP融合蛋白，测试D65-PLCG2是否表现出与PLCG2类似的Ca²⁺反应。
我们报告了PLCG2的多种亚型，包括（i）一种用来自相邻长链非编码（LNC）RNA（LINC00599）的序列替换外显子1的转录本，以及（ii）一种从外显子28起始处缺失65 bp的转录本。在人脑和血白细胞层样本中，PLCG2与经典PLCG2的比例均与rs12445675基因型相关。rs1071644（位于外显子28的65 bp可变剪接部分的一个T/C单核苷酸多态性）的T等位基因增加了以D65-PLCG2形式表达的PLCG2的比例。该单核苷酸多态性在小基因剪接试验中被证明具有功能。此外，发现rs1071644-T等位基因与AD风险增加相关，独立于rs72824905（P522R）和rs12445675。D65-PLCG2易受无义介导的RNA衰变影响。D65-PLCG2对Ca²⁺的反应方式与PLCG2不同。因此，rs1071644-T等位基因似乎通过增加以D65-PLCG2形式表达的PLCG2的比例来增加AD风险，这代表了PLCG2功能的丧失。
我们报告了两个AD遗传风险因素rs12445675和rs1071644，分别通过影响PLCG2与D65-PLCG2的比例和外显子28的剪接来影响AD风险。
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