Phys & Genetic Effects Of Disease-Causing Mutations of t

人促黄体激素/绒毛膜促性腺激素受体（LHR）的组成型激活突变导致家族性男性限制性早熟（FMPP），这是一种非中枢性促性腺激素非依赖性早熟。在Malcolm Martin博士和Ellen Leschek博士的合作下，我们确定了两名发生睾丸肿瘤的FMPP患者。为了研究LH/hCG信号通路的组成性激活对精子发生和性发育的影响，以及组成性激活的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。用携带激活突变的LHR转染MA-10细胞。使用cDNA微阵列将表达突变的LHR的细胞中的表达基因的谱与对照细胞的表达基因的谱进行比较。对一种突变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，已知参与精子发生的几个基因在表达突变LHR的细胞中也下调。这项研究正在重复与不同的突变LHR基因的分析，采用更广泛的小鼠cDNA微阵列。 与FMPP相反的是Leydig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，引起高促性腺激素性性腺功能减退症或男性假两性畸形。我们最近在一位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。T1505 C单碱基取代导致LHR跨膜螺旋（TM）IV中的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，突变型受体在hCG刺激下不能触发cAMP产生。TM IV在LHR信号转导中的作用尚不清楚。这种突变提供了一种工具，以调查的作用TM IV在活性-非活性构象转变的受体。已知突变的LHR，无论是激活还是失活，都被细胞异常加工。为了研究突变的LHR在体外的运输，我们将绿色荧光蛋白（GFP）的编码序列融合到野生型和突变的LHR的编码序列。将通过荧光显微镜研究融合蛋白的运输。产生的信息应该进一步我们的LHR的细胞处理的理解。 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在脑中的表达已被证实。我们推测FMPP患者的异常行为是由脑中突变的LHR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。为了实现这一目标，我们正在生成包含2 Kb 5？鼠LHR的UTR推定启动子序列和GFP偶联的野生型和突变的LHR，用于引入小鼠ES细胞以产生转基因动物。所产生的动物模型将用于研究组成性激活的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.