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）是指一个人出生时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个疾病基因与DSD相关，但目前大多数DSD患者即使在外显子组测序后也没有接受分子诊断。由于个体预后不确定，这代表了临床DSD管理的巨大挑战。我们认为，缺乏有关雄激素依赖性生殖器中线闭合程序中所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的病理学。在这里，我们假设，在DSD小鼠模型（TFM小鼠）的外生殖器结节中的单细胞转录组和染色质可及性的联合分析将揭示DSD发展和雄激素途径的调控结构的关键见解，并有助于识别新的疾病基因和与DSD相关的关键非编码元件。为了研究这一假设，我们将采用以下三种实验方法：目的1：在AR基因敲除小鼠（XY TFM）中产生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。目标二：创建可能与DSD相关的候选基因和非编码调控元件图谱目的3：通过基因组DNA测序，在从培养的人DSD患者外生殖器生殖器成纤维细胞建立的DNA样本中交叉验证来自开发的小鼠参考图谱的候选基因，其中患者的病理性APOD测定表明细胞雄激素信号传导的分子破坏。我们的研究将为DSD的发展产生新的分子见解，并确定有助于DSD的新疾病基因和非编码增强子元件。这些非编码调控元件中的突变原则上可以解释相当大比例的迄今未解决的DSD病例。

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.