Transcriptome profiling identifies regulators of pathogenesis in collagen VI related muscular dystrophy

ObjectivesThe collagen VI related muscular dystrophies (COL6-RD), Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are among the most common congenital muscular dystrophies and are characterized by distal joint laxity and a combination of distal and proximal joint contractures. Inheritance can be dominant negative (DN) or recessive depending on the type and location of the mutation. DN mutations allow incorporation of abnormal chains into secreted tetramers and are the most commonly identified mutation type in COL6-RD. Null alleles (nonsense, frameshift, and large deletions) do not allow incorporation of abnormal chains and act recessively. To better define the pathways disrupted by mutations in collagen VI, we have used a transcriptional profiling approach with RNA-Seq to identify differentially expressed genes in COL6-RD individuals from controls.MethodsRNA-Seq allows precise detection of all expressed transcripts in a sample and provides a tool for quantification of expression data on a genomic scale. We have used RNA-Seq to identify differentially expressed genes in cultured dermal fibroblasts from 13 COL6-RD individuals (8 dominant negative and 5 null) and 6 controls. To better assess the transcriptional changes induced by abnormal collagen VI in the extracellular matrix (ECM); we compared transcriptional profiles from subjects with DN mutations and subjects with null mutations to transcriptional profiles from controls.ResultsDifferentially expressed transcripts between COL6-RD and control fibroblasts include upregulation of ECM components and downregulation of factors controlling matrix remodeling and repair. DN and null samples are differentiated by downregulation of genes involved with DNA replication and repair in null samples.ConclusionsDifferentially expressed genes identified here may help identify new targets for development of therapies and biomarkers to assess the efficacy of treatments.研究目标：VI型胶原相关肌病（collagen VI related muscular dystrophies, COL6-RD）、乌里希先天性肌营养不良（Ullrich congenital muscular dystrophy, UCMD）与贝登肌病（Bethlem myopathy, BM）是最常见的先天性肌营养不良类型之一，其临床特征为远端关节松弛，同时合并远端与近端关节挛缩。该病的遗传模式可分为显性负性（dominant negative, DN）与隐性遗传两类，具体取决于突变的类型与位置。显性负性突变可使异常胶原链整合至分泌的四聚体中，是VI型胶原相关肌病中最常检出的突变类型。无效等位基因（null alleles，包括无义突变、移码突变及大片段缺失）无法使异常链完成整合，遗传方式呈隐性。为更清晰地阐明VI型胶原突变所扰乱的信号通路，本研究采用RNA测序（RNA-Seq）转录组分析方法，以鉴定VI型胶原相关肌病患者与健康对照之间的差异表达基因。 研究方法：RNA测序可精准检测样本中所有表达的转录本，并提供全基因组范围内的表达量量化工具。本研究对13名VI型胶原相关肌病患者（8例显性负性突变、5例无效等位基因突变）及6名健康对照的培养皮肤成纤维细胞进行RNA测序，以鉴定差异表达基因。为进一步评估细胞外基质（extracellular matrix, ECM）中异常VI型胶原诱导的转录变化，本研究分别将显性负性突变组与无效等位基因突变组的转录组特征与健康对照组进行对比。 研究结果：VI型胶原相关肌病患者与对照成纤维细胞间的差异表达转录本包括细胞外基质组分的上调，以及调控基质重塑与修复的因子的下调。显性负性突变组与无效等位基因突变组的差异则体现为：无效等位基因突变组中参与DNA复制与修复的基因表达显著下调。 研究结论：本研究鉴定出的差异表达基因可为新型治疗靶点开发以及评估治疗有效性的生物标志物研发提供潜在方向。