Integrative Approaches to Decipher Genetic Determinants of Disease Penetrance in Prokineticin 2 Pathway Related Human Reproductive Disorders

实现基因组精准医学的一个主要挑战是观察到基因型不 总是预测表型。不完全的表达和可变的表达是两个例子 表型变异性人类PROK 2（编码前动力蛋白）突变很好地说明了这种复杂性 2)和PROKR 2（促性腺激素受体2），导致人类分离的GnRH（促性腺激素释放激素） 缺乏症（IGD）。前动力蛋白2信号是哺乳动物生殖的主要调节因子， 与人类神经发育和神经内分泌形式的IGD（Kallmann综合征和正常嗅觉的IGD）相关。 IGD）。除了这种生理复杂性之外，PROK 2/R2突变主要是错义突变。 表现出降低的表达率和/或可变的表达率的突变。这种变异性的遗传基础 尚不完全清楚，但等位基因异质性和修饰基因已被假定为解释这一点 可变性在特定目标1中，将使用以下方法在体外检查人PROKR 2突变的等位基因异质性： PROK 2信号传导测定和等位基因特异性表达研究。全外显子测序和SNP基因分型 将确定修饰基因，有助于疾病的表达和表达。PROK 2还涉及到 不同的生理过程，如葡萄糖代谢、能量平衡、食物摄入、昼夜节律、疼痛 调节、中性粒细胞趋化性和血管生成。这些多效性功能的人类相关性 PROK 2尚未建立。在特定目标2中，PROK 2/R2突变的人类的表型解剖 将着手确定与人类PROK 2/R2突变相关的多效性表型。IGD患者 以及三级医院生物库群体中的对照个体。生殖表型 和非生殖表型将被绘制成图表。靶向表型分析将包括血糖评估 稳态和疼痛调节。一项全表型关联研究将在 携带PROK 2/R2突变的生物样本库受试者。在Specific Aim 3中，使用新的PROKR 2-cre小鼠模型， PROKR 2和GnRH神经元的转录组学研究将在不同的发育时间点进行， 使用化学遗传学暂时抑制PROKR 2。这些小鼠的性成熟和生育能力在时间上 还将检查PROKR 2抑制。在小鼠中差异表达的主要基因 转录组学实验将通过评估这些基因是否作为 人类全外显子组测序研究中的修饰基因。该提案中的研究有可能 为了揭示与PROK 2通路突变相关的疾病遗传的新机制， 与该途径中的突变相关的潜在多效性人类表型，并帮助提供个性化的 治疗生殖障碍和不育症的药物。

A major challenge in accomplishing genomic precision medicine is the observation that genotype does not always predict phenotype. Incomplete penetrance and variable expressivity represent two examples of this phenotypic variability. This complexity is well-illustrated by human mutations in PROK2 (encoding prokineticin 2) and PROKR2 (prokineticin receptor 2), that cause human Isolated GnRH (gonadotropin-releasing hormone) Deficiency (IGD). Prokineticin 2 signaling is a master regulator of mammalian reproduction and is associated with neurodevelopmental and neuroendocrine forms of IGD in humans (Kallmann syndrome, and normosmic IGD, respectively). In addition to this physiologic complexity, PROK2/R2 mutations are predominantly missense mutations that exhibit either reduced penetrance and/or variable expressivity. The genetic basis of this variability is not fully understood but allelic heterogeneity and modifier genes have been postulated to account for this variability. In Specific Aim 1, allelic heterogeneity of human PROKR2 mutations will be examined in vitro, using PROK2 signaling assays and allele specific expression studies. Whole exome sequencing and SNP genotyping will identify modifier genes that contribute to disease penetrance and expressivity. PROK2 is also implicated in diverse physiologic processes such as glucose metabolism, energy balance, food intake, circadian rhythms, pain modulation, neutrophil chemotaxis and angiogenesis. The human relevance of these pleotropic functions of PROK2 is yet to be established. In Specific Aim 2, phenomic dissection of humans with PROK2/R2 mutations will be undertaken to define the pleotropic phenotypes relating to PROK2/R2 mutations in humans. IGD patients as well as control individuals in a tertiary hospital biobank population will be studied. Reproductive phenotypes and non-reproductive phenotypes will be charted. Targeted phenotyping will include assessment of glucose homeostasis and pain modulation in both groups. A phenome-wide association study will be conducted in the biobank subjects harboring PROK2/R2 mutations. In Specific Aim 3, using a novel PROKR2-cre mouse model, transcriptomic studies of PROKR2 and GnRH neurons will be done at various developmental timepoints using temporal inhibition of PROKR2 using chemogenetics. Sexual maturation and fertility of these mice after temporal PROKR2 inhibition will also be examined. The top genes that are differentially expressed in the murine transcriptomic experiments will be translationally validated by assessing whether these genes emerge as modifier genes in the human whole exome sequencing studies. The studies in this proposal have the potential to unravel novel genetic mechanisms of disease penetrance relating to the PROK2 pathway mutations, uncover potential pleotropic human phenotypes associated with mutations in this pathway and help deliver personalized medicine to humans with reproductive disorders and infertility.