Downstream of the “Androgen receptor-bottleneck”: the transcriptional and chromatin landscape of urogenital midline fusion at single cell resolution and its relevance to differences of sex development (DSD)

性别发育差异（DSD）指的是一个人出生时的生殖或性解剖结构不符合女性或男性的典型定义。到目前为止，已有75种疾病基因与DSD相关，但目前大多数DSD患者即使在进行了外显子组测序后也没有得到分子诊断。由于个体预后不确定，这对临床dsd管理提出了巨大挑战。我们认为，缺少关于雄激素依赖性生殖器中线闭合程序中涉及的基因和途径的知识是导致这一问题的重要原因。单细胞基因组学领域的最新进展彻底改变了我们研究发育和疾病的方式，也有助于理解DSD的病理。在这里，我们假设对DSD小鼠模型（TFM小鼠）外生殖器结节中单细胞转录组和染色质可及性的联合分析将揭示DSD发展和雄激素通路调控结构的关键见解，并有助于识别与DSD相关的新疾病基因和关键非编码元件。为了研究这一假设，我们将采用以下三种实验方法：目的1：生成AR敲除小鼠（XY TFM）外生殖器发育和尿道管形成的联合单细胞转录组和染色质可及性图谱。目的2：建立候选基因图谱和可能与DSD相关的非编码调控元件。目的3：在人类DSD患者外生殖器培养的生殖成纤维细胞DNA样本中，通过与病理性APOD检测患者的基因组DNA测序，交叉验证已开发小鼠参考图谱中的候选基因。我们的研究将为DSD的发展提供新的分子见解，并确定新的疾病基因和促进DSD的非编码增强子元件。这些非编码调控元件的突变原则上可以解释迄今为止未解决的DSD病例的很大一部分。

Differences of sex development (DSD) refer to conditions in which a person is born with reproductive or sexual anatomy that doesn’t fit the typical definitions of female or male. So far 75 disease genes have been associated with DSDs, but currently the majority of patients with DSD do not receive a molecular diagnosis even after Exome sequencing. This represents a huge challenge in clinical DSD-management due to an uncertain individual prognosis. We assume that missing knowledge about the genes and pathways involved in the androgen dependent genital midline closure program contributes significantly to this problem. The recent advances in the field of single-cell genomics have revolutionized how we study development and disease and could also help understanding the pathology of DSD. Here we hypothesize that joint profiling of single-cell transcriptome and chromatin accessibility in the external genital tubercle of a mouse model for DSD (TFM mouse) will reveal critical insights into the development of DSD and the regulatory architecture of the androgen pathway and help identify new disease genes and critical non-coding elements related to DSD. To investigate this hypothesis, we will apply the following three experimental approaches: Objective 1: To generate a joint single-cell transcriptome and chromatin accessibility atlas of external genital development and urethral tube formation in AR knock-out mice (XY TFM). Objective 2: To create an atlas of candidate genes and non-coding regulatory elements potentially associated with DSD Objective 3: To cross-validate candidate genes from the developed mouse reference atlas in DNA-samples established from cultured genital fibroblasts derived from the external genitalia of human DSD-patients by genome DNA sequencing enriched with patients with a pathological APOD assay indicating molecular disruption of cellular androgen signaling. Our study will generate new molecular insights into the development of DSD and identify new disease genes and non-coding enhancer elements that contribute to DSD. Mutations in these non-coding regulatory elements could in principle explain a substantial proportion of so far unresolved cases of DSD.