脐带血蛋白质组与婴儿早期神经发育风险的关联

Association between umbilical cord blood proteome and early infant neurodevelop- mental risk

摘要

目的：利用前瞻性出生队列，通过高通量蛋白质组学技术，系统探讨脐带血蛋白质表达谱与婴儿早期神经心理发育的关联，识别潜在的早期生物标志物，并探索其背后的生物学机制，为在普通人群中早期识别神经发育风险、理解神经发育偏离的分子基础提供科学依据。方法：研究基于北京大学通州出生队列，纳入 96 例在1岁和3岁时完成《年龄与发育进程问卷（第三版）》（ages＆stages questionnaires，third edition，ASQ-3）评估的儿童。根据ASQ-3筛查结果将其分为异常组（n=42）和对照组（n=54）。检测并分析出生时冻存的脐带血血浆样本，采用非靶向定量蛋白质组学技术进行无偏倚篮查。通过差异表达分析、主成分分析（principal component analysis，PCA）、正交偏最小二乘判别分析（orthogonal partial least squares discriminant analysis，OPLS-DA）及加权基因共表达网络分析（weighted gene co-expression network analysis，WGCNA）等方法，识别差异表达蛋白质，并进行基因本体论（gene onto-logy，GO）功能富集与京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，计算差异倍数（fold change，FC）。统计学检验采用独立样本 t 检验，多重检验校正采用 Benjamini-Hochberg 法计算错误发现率（false discovery rate，FDR）。结果：蛋白质组学分析共鉴定出8214个共有蛋白质，与对照组比较，在异常组中385个为差异表达蛋白质（$\mathit{P}<0.05,\mid {\mathrm{l}\mathrm{o}\mathrm{g}}_{2}\mathrm{F}\mathrm{C}\mathrm{I}>0.585$），其中189个上调，196个下调。PCA 和 OPLS-DA 分析显示两组样本存在系统性蛋白质表达差异。WGCNA 分析鉴定出10个共表达模块，其中黄色模块与 ASQ-3 异常分组显著负相关（r=-0.233,P=0.024），粉色模块与沟通维度评分显著正相关（r=0.342,P=0.003）。富集分析表明，差异表达蛋白质及关键模块主要富集于两大功能通路：（1）遗传信息处理通路，包括核糖体、剪接体及 mRNA加工；（2）细胞骨架组织与 Wnt 信号通路，这些通路在神经发育障碍的发病机制中具有重要生物学意义。结论：脐带血中遗传信息处理及细胞骨架组织与 Wnt 信号通路相关蛋白质的扰动，可能是婴儿早期神经发育筛查异常的重要分子特征；本研究为在普通人群中早期识别神经发育风险提供了潜在的外周血生物标志物组合，并为理解神经发育偏离的生物学机制提供了新视角；未来需在更大规模队列中验证这些发现，并通过功能实验阐明关键蛋白质的具体作用机制。

Abstract

Objective：To systematically investigate the associations between umbilical cord blood pro- tein expression profiles and early infant neurodevelopment using a prospective birth cohort，to identify po- tential early biomarkers through high-throughput proteomics，and to explore underlying biological mecha- nisms，thereby providing scientific evidence for early identification of neurodevelopmental risks and un- derstanding the molecular basis of neurodevelopmental deviations in general populations. Methods： Based on the Peking University Birth Cohort in Tongzhou，this study enrolled 96 children who completed ages and stages questionnaires，third edition（ASQ-3）assessments at 1 and 3 years of age.Participants were classified into an abnormal group (n=42) and a control group (n=54) according to ASQ-3 screening results.Non-targeted quantitative proteomics was performed on cryopreserved umbilical cord blood plasma samples collected at birth.Differential expression analysis，principal component analysis （PCA），orthogonal partial least squares discriminant analysis（OPLS-DA），and weighted gene co-expression network analysis（WGCNA）were conducted to identify differentially expressed proteins，fol- lowed by Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）functional en- richment analyses.The fold change（FC）was calculated.Independent samples t-test was used for statis- tical comparison，with Benjamini-Hochberg method applied to calculate false discovery rate（FDR）for multiple testing correction. Results：Proteomic analysis identified 8214 common proteins，among which 385 proteins were differentially expressed（$\mathit{P}<0.05,\left|{\mathrm{l}\mathrm{o}\mathrm{g}}_{2}\mathrm{F}\mathrm{C}\right|>0.585$），including 189 proteins upre- gulated and 196 proteins downregulated in the abnormal group.PCA and OPLS-DA revealed systematic differences in protein expression patterns between the two groups.WGCNA identified 10 co-expression modules，with the yellow module showing significant negative correlation with ASQ-3 abnormal grouping（r= -0.233,P=0.024) and the pink module positively correlating with communication domain scores（r= 0.342,P=0.003）.Enrichment analyses demonstrated that differential proteins and key modules were primarily enriched in two functional categories：（1）genetic information processing pathways，including ribosome，spliceosome，and mRNA processing；and（2）cytoskeleton organization and Wnt signaling pathways.These pathways held significant biological relevance in the pathogenesis of neurodevelopmental disorders. Conclusion：Perturbations in proteins associated with genetic information processing and cy- toskeleton/Wnt signaling pathways in umbilical cord blood may represent important molecular characteris- tics of early neurodevelopmental screening abnormalities in infants.This study provides potential peri- pheral blood biomarker combinations for early identification of neurodevelopmental risks in general popu- lations and offers novel insights into the biological mechanisms underlying neurodevelopmental deviations. Future research should validate these findings in larger-scale cohorts and elucidate specific functional mechanisms of key proteins through experimental studies.

关键词

脐带血 / 蛋白质组学 / 神经发育 / 出生队列

Key words

Umbilical cord blood / Proteomics / Neurodevelopment / Birth cohort

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基金资助

国家自然科学基金(82204055)