Cologne Center for Genomics, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany; Institute of Human Genetics, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany; Institute of Human Genetics, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.
J Mol Diagn. 2022 Jun;24(6):619-631. doi: 10.1016/j.jmoldx.2022.02.006. Epub 2022 Apr 8.
Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging because of its high degree of sequence homology. Following first-line next-generation sequencing gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome and known SVs and 18 patients with atypical hemolytic uremic syndrome or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient, were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to next-generation sequencing was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease.
补体因子 H（CFH）及其相关蛋白在调节补体系统的替代途径中起着重要作用。CFH 基因簇（包括 CFH 及其五个相关基因 [CFHR1-5]）的突变和结构变异（SVs）已在肾脏病理学以及年龄相关性黄斑变性和系统性红斑狼疮等复杂免疫疾病中报道。由于其高度的序列同源性，该基因簇的 SV 分析具有挑战性。在进行了一线下一代测序基因面板测序后，我们应用 Genomic Vision 的 Molecular Combing Technology 来检测和可视化 CFH 基因簇内的 SVs，并完全解析其结构单倍型。该方法在三名具有非典型溶血性尿毒症综合征和已知 SVs 的患者以及 18 名具有非典型溶血性尿毒症综合征或补体因子 3 肾小球病且 CFH 基因簇单倍型未知的患者中进行了测试。在两名患者中发现了三种 SVs，一种是 CFH/CFHR1 杂交基因，在第三名患者中发现了一种罕见的杂合 CFHR4/CFHR1 缺失，与常见的 CFHR3/CFHR1 缺失发生在反位。使用针对长 DNA 片段的靶向富集方法（Samplix Xdrop）与牛津纳米孔测序相结合，确定了后者的断点。除了下一代测序之外，分子梳还能够提高该试点研究中的分子遗传学产量。这种（成本）有效的方法需要在具有 CFH/CFHR 相关疾病的更大队列中进行验证。
