Coding and non-coding genetic variants as the basis for early-onset osteoporosis

全基因组测序为全面检测个体变异提供了机会。然而，关于变异的潜在致病性的解释仍然是一个挑战，特别是在非编码基因组中。我们建议研究早发性骨质疏松症（EOOP）的遗传学，这是一种与常见疾病表型重叠的疾病。包含已知疾病基因的一组测序显示，400多名EOOP患者中约有20%发生了因果突变，表明这种疾病具有很强的遗传成分。我们在此提议对另外200例EOOP患者进行全基因组分析，其中排除了已知骨脆性疾病的基因突变。全基因组测序（WGS）数据将通过内部管道进行分析，以生成单核苷酸变异（snv）和结构变异（SVs）的综合列表。在这些患者的很大一部分中，我们期望非编码改变影响基因调控作为表型的基础，可能以寡生的方式。为了确定参与成骨细胞和破骨细胞（骨规则组）基因调控的基因组区域，我们将在间充质基质细胞（MSCs）、MSCs衍生的成骨细胞和来自健康供体的破骨细胞中使用染色质谱分析、ATAC-seq和RNA-seq。此外，我们将在这些细胞中执行染色体构象捕获（HiC）以生成全基因组相互作用谱。结合RNA-、ATAC-和ChIP-seq检测组蛋白标记和HiC，将使我们能够可靠地预测整个非编码基因调控元件及其相互作用域。这种联合方法将揭示骨质疏松GWAS研究中观察到的缺失遗传，并使EOOP患者分层和个性化治疗成为可能。

Whole genome sequencing offers the opportunity for a comprehensive detection of variants in an individual. However, the interpretation of variants in regard to their potential pathogenicity remains a challenge in particular in the non-coding genome. We propose to study the genetics of early-onset osteoporosis (EOOP), a condition with phenotypic overlap to common disease. Sequencing of a panel containing known disease genes revealed causal mutations in about 20% of more than 400 EOOP patients, showing that this condition has a strong genetic component. We here propose a genome-wide analysis of additional 200 EOOP patients in which mutations in genes for known bone fragility disorders have been excluded. Whole genome sequencing (WGS) data will be analysed with in-house pipelines to generate a comprehensive list of single nucleotide variants (SNVs) and structural variants (SVs). In a large part of these patients we expect non-coding alterations influencing gene regulation as the basis of the phenotype, possibly in an oligogenic manner. To identify the regions of the genome that are involved in gene regulation of osteoblasts and osteoclasts (bone regulome), we will use chromatin profiling, ATAC-seq, and RNA-seq in mesenchymal stromal cells (MSCs), MSC-derived osteoblasts and in osteoclasts from healthy donors. In addition, we will perform chromosome conformation capture (HiC) in these cells to generate genome-wide interaction profiles. The combination of RNA-, ATAC- and ChIP-seq for histone marks and HiC will enable us to reliably predict the entirety of non-coding gene regulatory elements and their interaction domains. This combined approach will shed light onto the missing inheritance observed in osteoporosis GWAS studies and enable stratification and individualized treatment of EOOP patients.