Purpose 50-60% of neuromuscular-disease patients remain undiagnosed even after extensive genetic testing that hinders precision-medicine/clinical-trial-enrollment. Importantly, those with DNA-based molecular diagnosis often remain without known molecular mechanism driving different degrees of disease severity that hinders patient stratification and trial-readiness. These are due to: a) clinical-genetic-heterogeneity (eg: limb-girdle-muscular-dystrophies(LGMDs)>30-subtypes); b) high-prevalence of variants-of-uncertain-significance (VUSs); (c) unresolved genotype-phenotype-correlations for patient stratification, and (d) lack of minimally-invasive biomarker-driven-assays. We therefore implemented a combinatorial phenotype-driven blood-biomarker functional-genomics approach to enhance diagnostics and trial-readiness by elucidating disease mechanisms of a neuromuscular-disease patient-cohort clinically-suspected of Dysferlinopathy/related-LGMD, the second-most-prevalent LGMD in the US. Methods We used CD14+monocyte protein-expression-assay on 364 Dysferlinopathy/related-LGMD-suspected patient-cohort without complete molecular-diagnosis or genotype-phenotype correlation; and then combined with blood-based targeted-transcriptome-sequencing (RNA-Seq) with tiered-analytical-algorithm correlating with clinical-measurements for a subset of patients. Results Our combinatorial-approach significantly increased the diagnostic-yield from 25% (N=326; 18%-27%; 95%CI) to 82% (N=38; 69.08% to 84.92%; 95% CI) by combining monocyte-assay with enhanced-RNA-Seq-analysis and clinical-correlation, following ACMG-AMP-guidelines. The tiered-analytical-approach detected aberrant-splicing, allele-expression-imbalance, nonsense-mediated-decay, and compound-heterozygosity without parental/offspring-DNA-testing, leading to VUS-reclassifications, identification of variant-pathomechanisms, and enhanced genotype-phenotype resolution including those with carrier-range Dysferlin-protein-expression and milder-symptoms, allowing patient-stratification for better trial-readiness. We identified uniform-distribution of pathogenic-variants across DYSF-gene-domains without any hotspot suggesting the relevance of upcoming gene-(full-DYSF-cDNA)-therapy trials. Conclusion Our results show the relevance of using a clinically-driven multi-tiered-approach utilizing a minimally-invasive biomarker-functional-genomic platform for precision-medicine-diagnostics, trial-recruitment/monitoring, elucidating pathogenic-mechanisms for patient stratification to enhance better trial outcomes, which in turn, will guide more rational use of current-therapeutics and development of novel-interventions for neuromuscular-disorders, and applicable to other genetic-disorders.

中文翻译：

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组合临床驱动的血液生物标志物功能基因组学显着增强了神经肌肉疾病的基因型-表型分辨率和诊断

目的即使在广泛的基因检测后，仍有50-60％的神经肌肉疾病患者仍无法诊断，这阻碍了精确医学/临床试验的招募。重要的是，那些具有基于DNA的分子诊断方法的人常常没有已知的分子机制来驱动不同程度的疾病严重程度，从而阻碍了患者的分层和试验准备。这些是由于：a）临床遗传异质性（例如：肢带-肌肉营养不良（LGMDs）> 30个亚型）；b）不确定性显着性变量（VUS）的普遍性；（c）用于患者分层的尚未解决的基因型-表型相关性，以及（d）缺乏微创生物标志物驱动的检测方法。因此，我们通过阐明临床上怀疑为神经纤维性疾病/相关性LGMD（第二高流行）的神经肌肉疾病患者队列的疾病机制，实施了组合表型驱动的血液生物标志物功能基因组学方法，以增强诊断和试验准备LGMD在美国。方法：我们对364例肾功能不全/相关的LGMD疑似患者进行了CD14 +单核细胞蛋白表达检测，但没有完整的分子诊断或基因型-表型相关性。然后将其与基于血液的靶向转录组测序（RNA-Seq）结合在一起，并与针对一部分患者的临床测量结果相关的分层分析算法。结果我们的组合方法将诊断产率从25％（N = 326; 18％-27％; 95％CI）显着提高到82％（N = 38; 69.08％增至84.92％; 遵循ACMG-AMP指南，将单核细胞分析与增强的RNA-Seq分析和临床相关性相结合，得出95％CI）。分层分析方法无需父母/后代DNA测试即可检测到异常剪接，等位基因表达失衡，无义介导的衰变和复合杂合性，从而导致VUS重新分类，变异致病机理鉴定和增强基因型-表型解析，包括那些携带者携带dysferlin蛋白表达和症状较轻的人，可将患者分层以更好地进行试验准备。我们确定了病原变体在DYSF基因域之间的均匀分布，没有任何热点，表明即将进行的基因（全DYSF-cDNA）治疗试验的相关性。