Neuromuscular and Rare Disease Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan.
Neurology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan.
Eur J Histochem. 2022 Sep 1;66(3):3418. doi: 10.4081/ejh.2022.3418.
Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies and four age-matched controls. Fluorescence data analysed in patients and compared to age-matched controls showed a significant reduction of the mutated sarcoglycan expression and a variable reduction of the other sarcoglycans. Fluorescence normalized data analysed in relation to the age of onset of the disease, showed a negative correlation of α-sarcoglycan fluorescent signal versus fibrosis in patients with an early age of onset and a negative correlation between δ-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful to better define both patient prognosis and expected disease evolution. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.
肌聚糖蛋白病在疾病进展、肌肉无力、丧失活动能力以及心脏/呼吸受累方面具有高度异质性。其临床严重程度通常与残留蛋白量相关，这使得蛋白定量极其重要。肌聚糖蛋白病的诊断是基于遗传学的，但骨骼肌分析——通过免疫组织化学和 Western blot（WB）——仍然是建立正确诊断过程的必要条件。然而，如果活检标本稀少，WB 分析则无法进行。本研究提供了一种基于骨骼肌切片免疫荧光染色、图像采集和软件处理的敏感工具，用于半定量检测肌聚糖蛋白病患者中肌聚糖的残留量。我们将这种方法应用于 11 例肌聚糖蛋白病、7 例贝克型肌营养不良症和 4 例年龄匹配的对照组。在患者中分析的荧光数据与年龄匹配的对照组进行比较，显示突变的肌聚糖表达显著减少，其他肌聚糖的表达也有不同程度的减少。将荧光归一化数据与疾病发病年龄相关分析，在发病年龄较早的患者中，α-肌聚糖荧光信号与纤维化呈负相关；在中晚期发病组中，δ-肌聚糖信号与纤维化呈负相关。这种能够客观定量肌细胞膜蛋白的方法的可用性，比 WB 分析更快、更节省，并且能够非常灵敏地检测到低残留的肌聚糖表达，这对于更好地定义患者的预后和预期疾病进展非常有用。该方法还可用于监测治疗干预的效果以及临床试验期间。
