Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α3α4α5(IV) and α1α1α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV):α1/2(IV) mean fluorescence intensities (α5:α1/2 intensity ratios) and thicknesses (α5:α1/2 thickness ratios) were calculated to represent the levels of collagen α3α4α5(IV) relative to α1α1α2(IV). The α5:α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic variants who could benefit from genetic testing.

中文翻译：

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局灶节段性肾小球硬化症和 Alport 基因变异患者石蜡切片中肾小球基底膜 IV 型胶原 α 链的定量评估

局灶节段性肾小球硬化症 (FSGS) 病变与基因变异有关，这些基因在阿尔波特综合征中也发生突变。尽管肾小球基底膜 (GBM) IV 型胶原 (colIV) 网络的定量评估可能有助于诊断，但并未广泛用于评估这些患者。为此，我们使用 Airyscan 共聚焦显微镜在肾脏石蜡切片上开发了胶原蛋白 α5(IV) 和 α1/2(IV) 的免疫荧光成像，可以清楚地将 GBM 胶原蛋白 α3α4α5(IV) 和 α1α1α2(IV) 区分为两个不同的层，从而可以定量对两个 colIV 网络的评估。计算胶原蛋白α5（IV）：α1/2（IV）平均荧光强度（α5：α1/2强度比）和厚度（α5：α1/2厚度比）的比率，以代表胶原蛋白α3α4α5（IV）的水平相对于α1α1α2(IV)。所有 11 个对照样本中的 α5:α1/2 强度和厚度比均具有可比性，而在所有具有致病性或可能致病性 Alport 变异的患者中，这两个比率均显着降低，支持了该方法的有效性。因此，随着这项技术的进一步验证，通过免疫荧光定量测量 GBM colIV 亚型丰度，可能有助于识别可能含有致病性变异的 FSGS 病变患者亚组，这些患者可以从基因检测中受益。