Phys & Genetic Effects Of Disease-Causing Mutations of t

人黄体生成素/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性早熟。在与Malcolm Martin博士和Ellen Leschek博士的合作中，我们已经确定了两名FMPP患者发展为睾丸肿瘤。为了研究LH/hCG信号通路组成性激活对精子发生和性发育的影响，以及组成性激活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。将携带激活突变的LHR转染MA-10细胞。利用cDNA微阵列技术将表达突变LHR的细胞与对照细胞的表达基因谱进行比较。对一个突变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LHR的细胞中，几个已知与精子发生有关的基因也被下调。利用更广泛的小鼠cDNA微阵列对不同突变的LHR基因进行了重复研究。

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.