不同位点NR5A1突变导致46，XY性发育异常表型异质性的机制研究

Disorders of Sexual Development（DSD）are rare congenital conditions in which there is divergence from the chromosome, gonad, or genitalia, classified as sex chromosome DSD , 46,XY DSD and 46, XX DSD. The etiology of 46, XY DSD is complex, with only 20% patients obtaining molecular diagnosis. Numerous mutations in genes involved in the androgen biosynthesis and actions pathway, as well as testis development have been disclosed to be the molecular basis of the disorders. During the testis formation from the primitive gonad, a crucial factor——SRY initiate the sex determination process and activate down-stream target gene, SOX9 (SRY-related HMG box gene). SOX9 up-regulates PGD2 and FGF9 genes, which maintain SOX9 expression, forming a positive feed-forward loop in XY gonads. NR5A1, expressed in the bipotential gonad of both genders, interact with SRY, WT1, GATA4 etc, playing a important role in the activation of SOX9. After asymmetric division of coelomic epithelium (multipotent progenitors）, SRY activating the expression of SOX9, pre-supporting cells differentiated as Sertoli cells, then testis develop gradually. Heterozygous mutation of NR5A1 was one of the leading cause of 46,XY DSD, but the phonotypical variation is tremendous and the inherited pattern remains unclear. In 46, XY subjects, NR5A1 heterozygous mutation could result in phenotype from gonad dysgenesis, presenting with hypospadias, microphallus and undescended testicle to male infertility, often without primary adrenocortical insufficiency. In our preliminary work, targeted next-generation sequencing was performed in the 70 46, XY DSD patients. Heterozygous mutation of NR5A1 was identified as the leading cause of the disorder (14.3%). Among 10 patients with NR5A1 mutation, patients showed different phenotypes, with 3 patients reared as female. AMH levels of all the patients was below the reference range, suggesting dysfunctional SC, while normal testosterone levels were identified in 5 patients (2 after hCG test), suggesting relatively normal function of Leydig cells. CYP17A1, marker of LC, was found to be reduced and even undetactable in the tesitis tissue by Immunohistochemistry, while the SOX9 staining of SC could be observed clearly. The missense mutaions (p.S430I, p.E367G, p.C370Y），frameshift variants （p.E148fsX295） and missense mutation p.C283* showed low expression level by Western Blot. Nine novel mutations of NR5A1 were found reducing the transcription activities of target gene（CYP11A1 and CYP19A1）, whereas p.T29K and p.N44del mutants were observed abnormal subnuclear structure under cofocal microscope. We assumed that different loci mutations of NR5A1, disturbing the differentiation and function of SC, and variable degrees of impairment of LC affect the androgen levels and virilization, leading to phenotype heterogeneity of the disorder. Future work consists in phenotype, hormone and histology analysis between the patients with different phenotypes (normal and low testosterone levels, testis dysgenesis and male infertility), inducing rescue expression of wild-type and mutant NR5A1 in NT2D1 cells and mLTC-1 cells with Cas9 KO endogenous NR5A1, observing the differentiation and function of SC and LC. Chip-seq and Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins (RIME) was performed to observe the different DNA combining sites and interactive proteins of WT and mutant NR5A1. Then T29K and G212S Knock-in mouse model will be constructed to investigate the influence of specific mutations on LC and SC differentiation and function in vivo. With the rapid development and mature of 3D cell culture technique, we are planning to establish the 3D culture system of testis organoids from testicular sperm extraction biopsies, which could be used in the orphan drug screening and reconstruction of the reproduction of patients.

46，XY性发育异常（46，XY DSD）是一种罕见病，导致严重的生殖发育障碍。课题组前期通过靶向二代测序发现NR5A1杂合突变是常见病因，但临床表型存在巨大异质性。由于致病机制和遗传规律不明使该疾病成为DSD诊治的难点。NR5A1在性腺两种体细胞Sertoli细胞（SC）和Leydig细胞（LC）分化和功能调节中起重要作用。根据患者表型、激素和性腺组织学检查发现NR5A1突变导致SC功能缺陷，而患者的表型异质性可能由不同突变对LC发育和功能影响不同所致。下一步拟通过在46，XY DSD和无精症样本中进行NR5A1突变筛查，深入分析表型和睾丸组织改变；在敲减NR5A1的NT2D1和mLTC-1细胞中用野生和不同位点突变的NR5A1进行rescue，进而通过Knock-in突变小鼠模型建立，观察不同突变对SC和LC分化及功能影响；并采用睾丸类器官3D培养，为孤儿药物筛查和生殖重建奠定基础。

46，XY性发育异常（46，XY DSD）是一类疾病的总称，导致严重的发育和生殖障碍。通过针对46，XY DSD的二代测序，在206例患者中明确分子诊断的有107例，其中NR5A1基因突变有19例。患者主要表现为不同程度的男性化不全，2例患者有脾脏发育异常；均无明显肾上腺皮质功能不全。19例患者中不同突变位点的患者临床表型具有较大的异质性，3例移码突变（p.E148fsX295、 p.Y404fs、p.R89fsX105）的患者外生殖器男性化严重缺陷；在10例错义突变的患者中，p.G35V和p.C370Y等表型相对较轻，而p.G212S和p.T29K等表型较为严重。 构建了6种新的突变体，报告基因检测发现不同突变位点对下游靶基因CYP11A1和CYP19A1等转录调控活性程度不同。p.Q42E和p.R84C突变部分影响NR5A1的核定位，移码突变p.L359_L363del导致核中出现异常聚集荧光信号。基于pLenti-CRISPRv2载体构建了针对NR5A1的序列的慢病毒质粒，建立敲除该基因的稳转细胞株；建立来源于患者iPSC的细胞模型，后续拟进行多能干细胞向Sertoli和Leydig细胞两种体细胞分化的研究。在基金的资助下，共发表SCI论文10篇: 1.对36例雄激素受体突变导致的46，XY DSD患者临床表型和突变功能研究，发现其中1例患者为NR5A1突变致病而非AIS；2.对中国人17β-HSD3缺陷导致的46，XY DSD的患者进行了临床特点以及突变酶功能研究；3. 对13例17-羟化酶/17,20裂解酶患者中，无高血压低血钾患者的CYP17A1的3种突变进行酶功能的研究，报道了2例患者成功生育，是全球的第4、5例；4.采用LC-MS/MS的方法，测定11-氧合类固醇激素有助于诊断表型重叠的AIS和5ɑ-还原酶缺陷；5. 在46，XY DSD患者的大家系中发现SRY R76L并非致病的原因，SOX9增强子的缺失可能是致病的原因；6.对特纳综合征患者的基因剂量进行的生物信息学分析；7.分离AIS患者的PBMC进行RNA-seq发现有免疫和代谢相关通路改变；8.对25例5ɑ-还原酶缺陷患者的分子病因的分析等。发表核心期刊3篇，建立靶向二代测序技术对206例46，XY DSD进行致病基因分布研究；对FGFR1突变导致卡尔曼综合特点和治疗转归分析