Supplementary file 2_Faecal inflammatory protein markers in children with autism spectrum disorder are comparable to their healthy siblings.docx

BackgroundAutism spectrum disorder (ASD) is a complex neurodevelopmental condition often accompanied by gastrointestinal (GI) symptoms. Inflammatory proteins in stool have been proposed as potential biomarkers, but evidence remains inconsistent. We compared fecal levels of α1-antitrypsin (A1AT), immunoglobulin A (IgA), and calprotectin (Cal) in 57 children with ASD and 57 biological siblings without ASD. Sibling designs are now preferred to disentangle ASD-specific biology from shared environmental and microbiome factors. Participants were carefully screened to exclude recent antibiotic use, digestive problems, gastrointestinal infections, and abnormal dietary patterns, thereby controlling for major factors known to influence gut inflammatory markers.
MethodsStool samples were thawed, freeze-dried, and proteins extracted using ammonium bicarbonate buffer with sodium deoxycholate. After BCA quantification, samples were reduced, alkylated, spiked with stable isotope–labelled peptides, and digested with trypsin. Peptides were purified and analyzed by UHPLC–MS/MS (Agilent 6495A) in dynamic SRM mode. Quantification used internal standards and normalization to total protein. Ratios of IgA1/IgA2 and S100A8/S100A9 were calculated. ASD severity was evaluated using the Childhood Autism Rating Scale (CARS).
ResultsChildren with ASD showed trends toward higher IgA and calprotectin and lower α1-antitrypsin compared with siblings, but differences were not statistically significant. Subgroup analysis suggested different distribution patterns in moderate versus severe ASD, including higher IgA in the moderate group and altered S100A8/S100A9 ratio in the severe group. These subgroup findings were exploratory, derived from critically underpowered post-hoc analyses (severe subgroup: n = 11 pairs, ~18% power for medium effects), and should be considered hypothesis-generating only, pending validation in adequately powered pre-registered studies.
ConclusionsThe results are consistent with recent meta-analyses reporting no consistent evidence of gut inflammation in ASD. Larger, sex-matched studies with full assay validation are needed to clarify the role of stool proteins in ASD.

## 背景
自闭症谱系障碍（Autism Spectrum Disorder, ASD）是一类复杂的神经发育疾病，常伴随胃肠道（GI）症状。粪便中的炎症蛋白曾被认为是潜在的生物标志物，但相关证据仍缺乏一致性。本研究对比了57名ASD患儿与57名无ASD的生物学同胞的粪便α1-抗胰蛋白酶（α1-antitrypsin, A1AT）、免疫球蛋白A（Immunoglobulin A, IgA）及钙卫蛋白（calprotectin, Cal）水平。同胞对照设计如今被优选用于区分ASD特异性生物学特征与共同环境及微生物组因素。研究对象均经过严格筛选，排除了近期使用抗生素、消化功能异常、胃肠道感染及异常饮食模式的情况，以此控制已知会影响肠道炎症标志物的主要混杂因素。

## 方法
粪便样本经解冻、冷冻干燥后，使用含脱氧胆酸钠的碳酸氢铵缓冲液提取蛋白。经BCA定量后，对样本进行还原、烷基化处理，加入稳定同位素标记肽段，再用胰蛋白酶进行酶解。肽段经纯化后，采用超高效液相色谱-串联质谱（UHPLC-MS/MS，安捷伦6495A）以动态选择反应监测（SRM）模式进行分析。定量采用内标法，并以总蛋白进行归一化处理。同时计算了IgA1/IgA2及S100A8/S100A9的比值。采用儿童自闭症评定量表（Childhood Autism Rating Scale, CARS）评估ASD严重程度。

## 结果
与同胞对照相比，ASD患儿的IgA及钙卫蛋白水平呈升高趋势，α1-抗胰蛋白酶水平呈降低趋势，但上述差异均无统计学意义。亚组分析显示，中度与重度ASD患儿存在不同的分布模式：中度ASD组的IgA水平更高，重度ASD组的S100A8/S100A9比值存在异常改变。上述亚组分析结果属于探索性分析，基于效力严重不足的事后检验（重度亚组：n=11对，中等效应量下统计效力约18%），仅可用于生成假说，需在效力充足的预先注册研究中完成验证后方可采信。

## 结论
本研究结果与近期荟萃分析结论一致，即未发现ASD患儿存在肠道炎症的一致性证据。未来需开展更大样本量、性别匹配且完成完整实验验证的研究，以明确粪便蛋白在ASD中的作用。