目的:确定新的生物标志物作为四肢带型肌营养不良(LGMD)的替代诊断工具。
背景:LGMD包括一组以近端肌肉无力为特征的肌营养不良,肌肉活检发现CK水平升高和营养不良。杂合CAPN3突变与常染色体显性LGMD-4相关,而双等位基因突变可导致常染色体隐性LGMD-1。诊断目前通常基于需要肌肉活检或血液检查的侵入性方法。在大多数情况下,肌肉活检的蛋白质印迹(WB)分析对于诊断至关重要。因为肌肉样品是目前唯一已知的表达全长CAPN3同工型的组织。
方法:我们分析了包含60名LGMD患者的队列中的CAPN3。选定的患者接受了完整的神经系统检查,肌电图,肌肉活检,和用于原代成纤维细胞分离的皮肤活检。通过WB分析在肌肉和皮肤组织中评估CAPN3的量。从肌肉中分离出的总RNA,成纤维细胞和尿液被处理,并采用cDNA进行定性分析。通过qRT-PCR研究CAPN3的表达。已经使用PyMOL对CAPN33D结构进行了可视化和分析。
结果:在我们的患者中,检测到7种不同的CAPN3突变,其中两个是小说。对来自成纤维细胞和尿液的CAPN3转录物进行测序后,我们令人惊讶地检测到不同的CAPN3同工型,包括全长转录物。我们发现成纤维细胞和肌肉组织的蛋白质水平相当;特别是,与两种组织的对照相比,携带新型CAPN3突变的患者的蛋白质减少了30%.
结论:我们的发现首次显示尿液和皮肤样本中存在CAPN3全长转录本。此外,我们展示了肌肉和皮肤样本之间惊人的可比CAPN3蛋白水平,因此,当分子诊断结果不确定时,我们可以假设使用皮肤活检和尿液样本作为另一种侵入性较小的方法来评估CAPN3的含量.
OBJECTIVE: To identify novel biomarkers as an alternative diagnostic tool for limb girdle muscular dystrophy (LGMD).
BACKGROUND: LGMD encompasses a group of muscular dystrophies characterized by proximal muscles weakness, elevated CK levels and dystrophic findings on muscle biopsy. Heterozygous CAPN3 mutations are associated with autosomal dominant LGMD-4, while biallelic mutations can cause autosomal recessive LGMD-1. Diagnosis is currently often based on invasive methods requiring muscle biopsy or blood tests. In most cases Western blotting (WB) analysis from muscle biopsy is essential for a diagnosis, as muscle samples are currently the only known tissues to express the full-length CAPN3 isoform.
METHODS: We analyzed CAPN3 in a cohort including 60 LGMD patients. Selected patients underwent a complete neurological examination, electromyography, muscle biopsy, and skin biopsies for primary fibroblasts isolation. The amount of CAPN3 was evaluated by WB analysis in muscle and skin tissues. The total RNA isolated from muscle, fibroblast and urine was processed, and cDNA was used for qualitative analysis. The expression of CAPN3 was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL.
RESULTS: Among our patients, seven different CAPN3 mutations were detected, of which two were novel. After sequencing CAPN3 transcripts from fibroblast and urine, we detected different CAPN3 isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel CAPN3 mutation showed a 30% reduction in protein compared to controls from both tissues.
CONCLUSIONS: Our findings showed for the first time the presence of the CAPN3 full-length transcript in urine and skin samples. Moreover, we demonstrated surprisingly comparable CAPN3 protein levels between muscle and skin samples, thus allowing us to hypothesize the use of skin biopsy and probably of urine samples as an alternative less invasive method to assess the amount of CAPN3 when molecular diagnosis turns out to be inconclusive.