P2RX2外显子2复杂插入缺失破坏剪接并降低P2X2输出：对DFNA41及可变表现度的意义

文献信息

中文摘要

背景： P2RX2编码ATP门控的P2X2受体，该受体与常染色体显性非综合征性听力损失41（DFNA41）相关。DFNA41的特征是进行性感音神经性听力损失和对声创伤的易感性增加。然而，该基因内截短或影响剪接的变异的具体表型后果仍知之甚少。
方法： 我们使用靶向听力损失基因panel并结合共分离分析评估了一个汉族家系。使用pcMINI-C小基因试验评估P2RX2外显子2中复杂插入缺失（NM_174873: c.278_288delinsACGTGAAGCCCCC）的剪接后果，随后在异源细胞中表达重建的异常异构体后进行RT-qPCR和Western blotting。
结果： 先证者患有早发性重度至极重度感音神经性听力损失，携带从父亲遗传的杂合P2RX2复杂插入缺失，以及母源遗传的致病性GJB2移码变异。小基因试验揭示隐蔽剪接使用，产生两个带有提前终止密码子的移码异构体。两个突变异构体均显示稳态mRNA降低和P2X2蛋白输出显著减少，与整体P2X2表达降低一致。
结论： 我们在功能上证实该外显子2复杂插入缺失破坏剪接并显著降低P2X2输出。这些发现支持功能丧失和单倍剂量不足的机制。明显的家系内变异性——表现为先证者的极重度早发性听力损失相较于杂合父亲较轻的表型——强调了P2RX2相关DFNA41特征性的可变表现度和不完全外显。尽管寡基因调控是一个合理的概念框架，但需要谨慎解释和未来独立验证。

英文摘要

BACKGROUND: P2RX2 encodes the ATP-gated P2X2 receptor associated with autosomal dominant non-syndromic hearing loss 41 (DFNA41). DFNA41 is characterized by progressive sensorineural hearing loss and an increased susceptibility to acoustic trauma. However, the specific phenotypic consequences of truncating or splice-altering variants within this gene remain poorly understood.
METHODS: We evaluated a Han Chinese family using a targeted hearing-loss gene panel with segregation analysis. Splicing consequences of a complex indel in P2RX2 exon 2 (NM_174873: c.278_288delinsACGTGAAGCCCCC) were assessed using a pcMINI-C minigene assay, followed by RT-qPCR and Western blotting after expression of the reconstructed aberrant isoforms in heterologous cells.
RESULTS: The proband had early-onset severe-to-profound sensorineural hearing loss and carried a heterozygous P2RX2 complex indel inherited from the father, together with a maternally inherited pathogenic GJB2 frameshift. Minigene assays revealed cryptic splice usage that generated two frameshifted isoforms with premature termination codons. Both mutant isoforms showed reduced steady-state mRNA and markedly reduced P2X2 protein output, consistent with decreased overall P2X2 expression.
CONCLUSION: We functionally establish that this exon-2 complex indel disrupts splicing and markedly reduces P2X2 output. These findings support a mechanism of loss-of-function and haploinsufficiency. The pronounced intrafamilial variability-manifesting as profound early-onset hearing loss in the proband compared to a milder phenotype in the heterozygous father-underscores the variable expressivity and incomplete penetrance characteristic of P2RX2-linked DFNA41. Although oligogenic modulation is a plausible conceptual framework, it requires cautious interpretation and independent future validation.