Phys & Genetic Effects Of Disease-Causing Mutations of t

物理

• 批准号: 6664193
• 负责人: Owen M Rennert
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6664193
• 关键词: biological signal transduction; chorionic gonadotropin; complementary DNA; developmental genetics; gene induction /repression; gene mutation; genetically modified animals; hormone receptor; hormone regulation /control mechanism; laboratory mouse; luteinizing hormone; male; microarray technology; precocious puberty; spermatogenesis

Constitutive activating mutations of the human luteinizing hormone/chorionic gonadotropin receptor (LHR) cause familial male-limited precocious puberty (FMPP), a non-central form of gonadotropin-independent precocious puberty. In collaboration with Dr. Malcolm Martin and Dr. Ellen Leschek, we have identified two FMPP patients who developed testicular neoplasia. To study the impact of constitutive activation of the LH/hCG signaling pathway on spermatogenesis and sexual development, and the potential tumorigenic effect of a constitutively activated LHR, we have generated an in vitro cell model and are in the process of generating a transgenic animal model. MA-10 cells were transfected with LHR carrying activating mutations. The profile of expressed genes in cells expressing the mutated LHR was compared with that of control cells using cDNA microarrays. Preliminary studies of one mutated LHR indicated up-regulation of genes associated with cell proliferation and down-regulation of genes associated with differentiation. Interestingly, several genes known to be involved in spermatogenesis were also down-regulated in cells expressing the mutated LHR. This study is being repeated with different mutated LHR genes with analysis employing a more extensive mouse cDNA microarray. The antithesis of FMPP is Leydig Cell Hypoplasia (LCH). In LCH patients, mutation inactivates the LHR resulting in reduced production of testosterone causing hypergonadotrophic hypogonadism or male pseudohermaphroditism. We recently identified a novel inactivating homozygous mutation in the LHR of a 19-year old patient with male pseudohermaphroditism. The single base substitution T1505C caused the replacement of Leu-502 by Pro in transmembrane helix (TM) IV of the LHR. This change presumably disrupted the alpha helical structure of TM IV resulting in the inactivation of the LHR. This is the first disease-causing mutation identified in TM IV of the LHR. The mutant receptor failed to trigger cAMP production upon hCG stimulation in transient expression study. The role of TM IV in signal transduction of the LHR is not known. This mutation provides a tool to investigate the role of the TM IV in the active-inactive conformation transition of the receptor. It is known that the mutated LHR, be it activated or inactivated, are abnormally processed by cells. To investigate the trafficking of mutated LHR in vitro, we have fused the coding sequence of Green Fluorescent Protein (GFP) to that of wild- type and mutated LHR. The trafficking of the fused protein will be studied by fluorescent microscopy. Information generated should further our understanding of the cellular processing of the LHR. The impact of activating mutation of the LHR has always been considered to be limited to sexual development of the patient. The abnormal social behavior of the patient was thought to be secondary to precocious sexual maturation. Expression of the LHR in the brain had been demonstrated. We speculate that the abnormal behavior of FMPP patients is caused by the expression of the mutated LHR in the brain. The first step to examine this hypothesis is to identify the cellular location of the LHR in the brain. In order to achieve this, we are generating constructs containing a 2 Kb 5?UTR putative promoter sequence of murine LHR and a GFP-coupled wild-type and mutated LHR for introduction into mouse ES cells to generate the transgenic animals. The animal model generated will be used to study the impact of constitutively activated LHR on spermatogenesis as well as sexual and neurological development.

人类黄体生成素/绒毛膜促性腺激素受体（LHR）的组成性激活突变导致家族性男性局限性性早熟（FMPP），这是一种非促性腺激素依赖性性早熟的非中心形式。我们与 Malcolm Martin 博士和 Ellen Leschek 博士合作，确定了两名患有睾丸肿瘤的 FMPP 患者。为了研究LH/hCG信号通路的组成型激活对精子发生和性发育的影响，以及组成型激活的LHR的潜在致瘤作用，我们已经建立了体外细胞模型，并正在建立转基因动物模型。 MA-10 细胞被携带激活突变的 LHR 转染。使用cDNA微阵列将表达突变LHR的细胞中表达基因的谱与对照细胞的表达谱进行比较。对一种突变的 LHR 的初步研究表明，与细胞增殖相关的基因上调，而与分化相关的基因下调。有趣的是，一些已知参与精子发生的基因在表达突变 LHR 的细胞中也被下调。这项研究正在用不同的突变 LHR 基因进行重复，并使用更广泛的小鼠 cDNA 微阵列进行分析。 FMPP 的对立面是间质细胞发育不全 (LCH)。在 LCH 患者中，突变使 LHR 失活，导致睾酮产生减少，导致促性腺激素亢进性性腺功能减退症或男性假两性畸形。我们最近在一名 19 岁男性假两性畸形患者的 LHR 中发现了一种新的失活纯合突变。单碱基取代 T1505C 导致 LHR 的跨膜螺旋 (TM) IV 中 Leu-502 被 Pro 取代。这种变化可能破坏了 TM IV 的 α 螺旋结构，导致 LHR 失活。这是 LHR TM IV 中发现的第一个致病突变。在瞬时表达研究中，突变受体在 hCG 刺激下未能触发 cAMP 产生。 TM IV 在 LHR 信号转导中的作用尚不清楚。该突变提供了一种工具来研究 TM IV 在受体活性-非活性构象转变中的作用。众所周知，突变的LHR，无论是激活的还是失活的，都会被细胞异常处理。为了研究突变 LHR 的体外运输，我们将绿色荧光蛋白 (GFP) 的编码序列与野生型和突变 LHR 的编码序列融合。将通过荧光显微镜研究融合蛋白的运输。生成的信息应该可以进一步加深我们对 LHR 细胞处理的理解。 LHR 激活突变的影响一直被认为仅限于患者的性发育。患者的异常社会行为被认为是继发于性早熟。 LHR 在大脑中的表达已得到证实。我们推测FMPP患者的异常行为是由大脑中突变的LHR表达引起的。检验这一假设的第一步是确定 LHR 在大脑中的细胞位置。为了实现这一目标，我们正在生成含有鼠LHR的2Kb 5UTR推定启动子序列和GFP偶联的野生型和突变LHR的构建体，用于引入小鼠ES细胞以产生转基因动物。生成的动物模型将用于研究组成型激活的 LHR 对精子发生以及性和神经发育的影响。