背景:我们旨在分析Duchenne型肌营养不良(DMD)和Becker型肌营养不良(BMD)患者之间的全基因组转录组差异,并确定与肌肉磁共振成像(MRI)和组织学纤维脂肪置换相关的生物标志物。尚未报告。
方法:纳入了101例男性肌萎缩蛋白病(55DMD和46BMD)患者。在31例DMD患者中进行了肌肉来源的全基因组RNA测序,29例BMD患者,11个正常对照。在所有患者的肌肉MRI和组织学水平上对纤维脂肪替代进行评分。一条独特的管道,单样本基因集富集分析结合Spearman等级相关性(ssGSEA-Cor)被开发用于鉴定纤维脂肪替代的最相关的基因特征。实时定量PCR(qRT-PCR)分析,蛋白质印迹分析,对其余患者进行了单核RNA测序(snRNA-seq),以验证最相关的基因特征.
结果:比较转录组学分析显示,与29块BMD肌肉相比,31块DMD肌肉的炎症/免疫反应和细胞外基质重塑显著增加(P<0.05)。ssGSEA-Cor管道显示,CDKN2A和CDKN2B的基因集是纤维脂肪替代治疗中最相关的基因标记(组织学rs=0.744,P<0.001;MRIrs=0.718,P<0.001)。肌肉qRT-PCR证实15例DMD(中位数=25.007,P<0.001)和12例BMD(中位数=5.654,P<0.001)患者的CDKN2AmRNA表达均显著高于对照组(中位数=1.101),而CDKN2BmRNA表达在DMD间无显著差异,BMD,和对照组。在27名患者中,肌肉CDKN2AmRNA表达分别与肌肉MRI(rs=0.883,P<0.001)和组织学纤维脂肪替代(rs=0.804,P<0.001),疾病持续时间(rs=0.645,P<0.001)和NorthStar门诊评估总分(rs=-0.698,P<0.001)相关。肌肉免疫印迹分析证实4例DMD(中位数=2.958,P<0.05)和4例BMD(中位数=1.959,P<0.01)患者的CDKN2A蛋白表达水平明显高于对照组(中位数=1.068)。两个DMD肌肉的snRNA-seq分析表明,CDKN2A主要在纤维脂肪原祖细胞中表达,卫星细胞,和成肌细胞.
结论:我们确定CDKN2A表达是纤维脂肪替代的新生物标志物,这可能是肌萎缩蛋白病抗纤维化治疗的新靶点。
BACKGROUND: We aimed to analyse genome-wide transcriptome differences between Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients and identify biomarkers that correlate well with muscle magnetic resonance imaging (MRI) and histological fibrofatty replacement in both patients, which have not been reported.
METHODS: One hundred and one male patients with dystrophinopathies (55 DMD and 46 BMD) were enrolled. Muscle-derived genome-wide RNA-sequencing was performed in 31 DMD patients, 29 BMD patients, and 11 normal controls. Fibrofatty replacement was scored on muscle MRI and histological levels in all patients. A unique pipeline, single-sample gene set enrichment analysis combined with Spearman\'s rank correlations (ssGSEA-Cor) was developed to identify the most correlated gene signature for fibrofatty replacement. Quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, and single-nucleus RNA-sequencing (snRNA-seq) were performed in the remaining patients to validate the most correlated gene signature.
RESULTS: Comparative transcriptomic analysis revealed that 31 DMD muscles were characterized by a significant increase of inflammation/immune response and extracellular matrix remodelling compared with 29 BMD muscles (P < 0.05). The ssGSEA-Cor pipeline revealed that the gene set of CDKN2A and CDKN2B was the most correlated gene signature for fibrofatty replacement (histological rs = 0.744, P < 0.001; MRI rs = 0.718, P < 0.001). Muscle qRT-PCR confirmed that CDKN2A mRNA expression in both 15 DMD (median = 25.007, P < 0.001) and 12 BMD (median = 5.654, P < 0.001) patients were significantly higher than that in controls (median = 1.101), while no significant difference in CDKN2B mRNA expression was found among DMD, BMD, and control groups. In the 27 patients, muscle CDKN2A mRNA expression respectively correlated with muscle MRI (rs = 0.883, P < 0.001) and histological fibrofatty replacement (rs = 0.804, P < 0.001) and disease duration (rs = 0.645, P < 0.001) and North Star Ambulatory Assessment total scores (rs = -0.698, P < 0.001). Muscle western blot analysis confirmed that both four DMD (median = 2.958, P < 0.05) and four BMD (median = 1.959, P < 0.01) patients had a significantly higher level of CDKN2A protein expression than controls (median = 1.068). The snRNA-seq analysis of two DMD muscles revealed that CDKN2A was mainly expressed in fibro-adipogenic progenitors, satellite cells, and myoblasts.
CONCLUSIONS: We identify CDKN2A expression as a novel biomarker of fibrofatty replacement, which might be a new target for antifibrotic therapy in dystrophinopathies.