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（hLHR）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。尽管尚不清楚FMPP患者中睾酮的组成性产生是否具有致瘤性，但我们已经确定了两名发生睾丸瘤变的FMPP患者。另一项睾丸肿瘤研究也在睾丸肿瘤患者亚组中发现了hLHR的激活突变。我们推测，激活突变的hLHR开启了最终导致睾丸肿瘤发展的基因。为了研究组成性激活的LHR的潜在致瘤作用，我们已经产生了体外细胞模型。用携带激活突变的hLHR转染小鼠Leydig细胞系MA-10细胞。使用NIH小鼠23 K基因的cDNA微阵列将表达突变hLHR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。在22，684个基因中，10，353个在包括MA 10（对照）和实验样品的所有微阵列图像中显示高质量的信息表达数据，包括正向和反向标记。最常见的激活突变Asp 578 Gly诱导了42个差异表达基因，包括Bscl 2、Tcfe 2a、Ercc 3、Crsp 9、Gabarapl 1、Il 10 rb和Hsh 2。在睾丸肿瘤患者中发现的体细胞激活突变Asp 578 His诱导了74个差异表达基因。许多基因在两个治疗组之间是共同的，包括Tcfe 2a、Crsp 9和Rpo 1 -2。所有这三个基因都参与转录。它们与突变的hLHR的关系将被进一步研究。 hLHR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在脑组织中的表达已被证实。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。为了验证这一假设，并研究组成性激活的LHR对精子发生以及性和神经发育的影响，我们已经产生了一种转基因小鼠品系，该品系表达增强型绿色荧光蛋白（EGFP）和Asp 578 Gly或Asp 578 His取代的hLHR突变体的融合蛋白。有趣的是，所有表达突变hLHR的转基因小鼠都是雌性。这种现象的原因尚不清楚。我们目前正在研究突变受体对胚胎生长和发育的影响。 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。一种新的错义突变A340 T导致Ile-114被Phe取代，其影响hLHR胞外结构域中的一个富含亮氨酸的重复序列（LRR），已在LCH患者中被鉴定。在瞬时表达研究中，突变型受体在hCG刺激后不能触发cAMP产生。该突变显然不影响突变受体的运输，如受体与绿色荧光蛋白的融合蛋白的荧光显微镜研究所揭示的。相反，它影响受体对激素的结合。生成LRR的计算机模型以研究这种突变对受体构象的影响。该模型清楚地表明了突变的构象效应。这一发现可以扩展到解释突变对其他具有LRR的蛋白质的生物活性的影响。

Constitutive activating mutations of the human luteinizing hormone/chorionic gonadotropin receptor (hLHR) cause familial male-limited precocious puberty (FMPP), a non-central form of gonadotropin-independent precocious puberty. Even though constitutive production of testosterone, which occurs in FMPP patients, is not known to be tumorigenic, we had identified two FMPP patients who developed testicular neoplasia. Another study of testicular tumor also identified an activating mutation of the hLHR in a subgroup of testicular tumor patients. We hypothesize that hLHR with activating mutation turns on genes that ultimately lead to the development of testicular tumor. To study the potential tumorigenic effect of a constitutively activated LHR, we have generated an in vitro cell model. MA-10 cells, a mouse Leydig cell line, were transfected with hLHR carrying activating mutations. The profile of expressed genes in cells expressing the mutated hLHR was compared with that of control cells using cDNA microarrays of the NIH mouse 23K genes. Out of 22,684 genes, 10,353 display high quality informative expression data across all microarray images including MA10 (control) and the experimental samples, in both forward and reverse labeling. The most common activating mutation Asp578Gly induces 42 differentially expressed genes including Bscl2, Tcfe2a, Ercc3, Crsp9, Gabarapl1, Il10rb, and Hsh2. The somatic activating mutation Asp578His found in some patients with testicular tumors induce 74 differentially expressed genes. A number of genes are common between the two treatment groups, including Tcfe2a, Crsp9, and Rpo1-2. All these three genes are involved in transcription. Their relationship with mutated hLHR will be further investigated. The impact of activating mutation of the hLHR 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 LHR in 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. To examine this hypothesis and to study the impact of constitutively activated LHR on spermatogenesis as well as sexual and neurological development, we have generated a transgenic mouse strain that expresses a fusion protein of enhanced green fluorescent protein (EGFP) and hLHR mutant with substitution Asp578Gly or Asp578His. Interestingly, all of the transgenic mice expressing the mutated hLHR are females. The reason for this phenomenon is unclear. We are currently investigating the impact of the mutated receptor on embryonic growth and development. 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. A novel missense mutation A340T resulting in the substitution of Ile-114 by Phe, which affects one of the Leucine-rich repeats (LRR) in the extracellular domain of the hLHR, has been identified in a patient with LCH. The mutant receptor fails to trigger cAMP production upon hCG stimulation in transient expression studies. This mutation apparently does not affect trafficking of the mutated receptor as revealed by fluorescent microscopic study of the fusion protein of receptor with green fluorescent protein. Instead, it affects binding of the hormone by the receptor. A computer model of the LRR is generated to study the effect of this mutation on the conformation of the receptor. The model clearly demonstrates the conformational effect of the mutations. This finding may be extended to explain the impact of mutations on the biological activities of other proteins with LRRs.