Phys & Genetic Effects Of Disease-Causing Mutations of t

人类黄体生成素/绒毛膜促性腺激素受体(LHR)的结构性激活突变会导致家族性男性限制性性早熟(FMPP)，这是一种非中枢形式的促性腺激素非依赖性性早熟。在马尔科姆·马丁博士和艾伦·莱斯切克博士的合作下，我们已经确定了两名FMPP患者发展为睾丸肿瘤。为了研究促黄体生成素/人绒毛膜促性腺激素信号通路的结构性激活对精子发生和性发育的影响，以及结构性激活的LHR的潜在致瘤作用，我们建立了一个体外细胞模型，并正在建立转基因动物模型。将携带激活突变的LHR基因导入MA-10细胞。用基因芯片技术比较表达突变的LHR细胞和对照细胞的基因表达谱。对一个突变的LHR的初步研究表明，与细胞增殖相关的基因上调，与分化相关的基因下调。有趣的是，在表达突变的LHR的细胞中，几个已知参与精子发生的基因也下调了。这项研究正在对不同突变的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良(LCH)。在LCH患者中，突变使LHR失活，导致睾酮产生减少，导致高促性腺激素减退症或男性假两性畸形。我们最近在一例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子突变。单碱基替换T1505C导致LHR跨膜螺旋(TM)IV中的Leu-502被Pro取代。这一变化可能破坏了TM IV的α螺旋结构，导致LHR失活。这是在LHR的TM IV中发现的第一个致病突变。在瞬时表达研究中，突变型受体未能在hCG刺激下触发cAMP的产生。TM IV在LHR信号转导中的作用尚不清楚。这一突变为研究TM IV在受体活性-非活性构象转变中的作用提供了一种工具。众所周知，突变的LHR，无论是激活的还是灭活的，都是由细胞异常处理的。为了研究突变的LHR的体外转运，我们将绿色荧光蛋白(GFP)的编码序列与野生型和突变的LHR的编码序列进行了融合。融合蛋白的运输将用荧光显微镜进行研究。产生的信息应该会进一步加深我们对LHR细胞处理过程的理解。 激活LHR突变的影响一直被认为仅限于患者的性发育。患者的异常社交行为被认为是性早熟的次要原因。LHR在脑内的表达已被证实。我们推测FMPP患者的异常行为是由突变的LHR在脑内的表达引起的。检验这一假设的第一步是确定LHR在大脑中的细胞位置。为了实现这一点，我们正在构建包含一个2kb的5‘非编码区的小鼠LHR的启动子序列和一个GFP偶联的野生型和突变的LHR的载体，用于导入小鼠ES细胞以产生转基因动物。所建立的动物模型将用于研究结构性激活的LHR对精子发生以及性和神经发育的影响。

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.