BACKGROUND Phospholipase Cγ2 (PLCγ2) is an important signaling molecule that receives and transmits signals from various cell surface receptors in most hematopoietic lineages. Variants of PLCG2 cause PLCγ2-associated immune dysregulation (PLAID), a family of conditions that are classified by mutational effect. PLAID with cold urticaria (PLAID-CU) is caused by in-frame deletions of PLCG2 that are dominant negative at physiologic temperatures but become spontaneously active at sub-physiologic temperatures. OBJECTIVE To identify genetic lesions that cause PLAID by combining RNA sequencing of full-length PLCG2 with whole genome sequencing. METHODS We studied nine probands with antibody deficiency and a positive evaporative cooling test, together with two known PLAID-CU patients and three healthy subjects. Illumina sequencing was performed on full-length PLCG2 cDNA synthesized from peripheral blood mononuclear cell RNA and whole genome sequencing was used to identify genetic lesions. Novel alternate transcripts were overexpressed in the Plcg2-deficient DT40 cell overexpression system. ERK phosphorylation was quantified by flow cytometry with and without BCR crosslinking. RESULTS Two probands expressed novel alternative transcripts of PLCG2 with in-frame deletions. Proband 1, expressing PLCG2 without exons 18-19, carried a splice site mutation in intron 19. Proband 2, expressing PLCG2 without exons 19-22, carried a 14kb de novo deletion of PLCG2. DT40 cells overexpressing the exon 18-19 or exon 19-22 deletions failed to phosphorylate ERK in response to BCR crosslinking. CONCLUSION In addition to autosomal dominant genomic deletions, de novo deletions and splice site mutations of PLCG2 can also cause PLAID-CU. All of these can be identified by cDNA-based sequencing.

中文翻译：

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剪接位点和从头变异可导致 PLCG2 相关免疫失调伴寒冷性荨麻疹 （PLAID-CU）。


背景磷脂酶 Cγ2 （PLCγ2） 是一种重要的信号分子，可接收和传递来自大多数造血谱系中各种细胞表面受体的信号。PLCG2 的变体会导致 PLCγ2 相关免疫失调 （PLAID），这是一类按突变效应分类的疾病。PLAID 伴寒冷性荨麻疹 （PLAID-CU） 是由 PLCG2 框内缺失引起的，这些缺失在生理温度下显性阴性，但在亚生理温度下自发活跃。目的 将全长 PLCG2 的 RNA 测序与全基因组测序相结合，确定导致 PLAID 的遗传病灶。方法 我们研究了 9 例抗体缺乏且蒸发降温试验阳性的先证者，以及 2 例已知的 PLAID-CU 患者和 3 例健康受试者。对外周血单核细胞 RNA 合成的全长 PLCG2 cDNA 进行 Illumina 测序，采用全基因组测序鉴定遗传病灶。在 Plcg2 缺陷型 DT40 细胞过表达系统中过表达新的替代转录本。通过流式细胞术定量 ERK 磷酸化，有和没有 BCR 交联。结果 两个先证者表达了 PLCG2 的新型替代转录本，但框内缺失。前证者 1 表达 PLCG2，无外显子 18-19，在内含子 19 中携带剪接位点突变。表达 PLCG2 的先证者 2 没有外显子 19-22，携带 14 kb 的 PLCG2 从头缺失。过表达外显子 18-19 或外显子 19-22 缺失的 DT40 细胞在 BCR 交联后未能磷酸化 ERK。结论 除常染色体显性遗传基因组缺失外，PLCG2 的从头缺失和剪接位点突变也可导致 PLAID-CU。所有这些都可以通过基于 cDNA 的测序来识别。