血清素是人體內主要的神經傳導物質之一，它與許多生理功能的調控有著密不可分的關係，例如:情緒反應、睡眠與體溫調節。當血清素傳遞功能失常時，可能會引發躁鬱症或經前症候群等不同程度的心理及生理問題。血清素受體1A (Serotonin receptor 1A ; HTR1A)已知是血清素傳遞系統中一個重要的自體受體 (autoreceptor) ，先前的研究指出血清素受體1A的功能失調和人類許多疾病，如精神病有關。在一個臨床病例中，有位女性病人其生理期即有反覆性的發燒，其家屬則另有精神性問題、偏頭痛、糖尿病等多種臨床表現。此病人的週期性發燒可藉由buspirone這個藥物有效控制，由於buspirone是血清素受體1A的促效藥，這也暗示了這個病人可能是由於體內缺乏血清素受體1A而造成血清素傳遞功能失常。本研究的目的是去探討血清素受體1A基因與此病人症狀的關係並研究潛在的分子機轉。我們先針對此病人及其家屬的血清素受體1A基因去做突變檢測並發現位於啟動子區的-480delA突變。這結果顯示血清素受體1A啟動子區的-480delA突變可能會改變基因本身與轉錄因子之間的作用進而影響基因的表現及功能。雙重螢光素酶報導基因檢驗(dual-luciferase reporter assay)的結果證實血清素受體1A的-480delA突變的確會降低其基因表現。而凝膠電泳位移測定(Electrophoretic mobility shift assay)也發現有多種轉錄因子可專一性的結合至血清素受體1A的-480啟動子區域。利用biotin pull-down assay及質譜儀分析，我們成功的找出poly(ADP-ribose) polymerase (PARP-1)可能為其中之一的轉錄因子。接著利用染色質免疫沉降法(chromatin immunoprecipitation assay)更進一步的確認了PARP-1與血清素受體1A啟動子之間的結合能力。此外，過度及降低PARP-1在細胞中的表現都相對影響血清素受體1A的表現量，因此證實PARP-1扮演血清素受體1A基因表現的抑制蛋白的角色。綜合以上結果，我們發現血清素受體1A的-480delA突變會導致較多的PARP-1結合至啟動子區，進而造成較明顯的基因抑制效果。而血清素受體1A基因的表現一旦減少可能會使得其在調節降低體溫的功能失衡，而造成我們所觀察到的臨床症狀。由於血清素受體1A是血清素傳遞過程重要的自體受體，與調節細胞間血清素的量及維持血清素傳遞平衡有關，本研究結果將是研究血清素相關疾病病理機制的珍貴資訊。

Serotonin (5-hydroxytryptamine) is one of the key neurotransmitters that involves in the regulation of many important physiological conditions, such as mood and thermoregulation. Dysfunction of serotonergic transmission has been implicated in the pathogenesis of many diseases, for example, bipolar disorder and premenstrual syndrome. Serotonin receptor 1A (HTR1A) is one of the autoreceptors for serotonin transmission. Dysfunction of HTR1A has been found to be associated with many human diseases such as psychosis. In clinic, we identified one 33-year-old female with periodic menstrual fever responded well to buspirone, a HTR1A agonist, it suggested that her periodic menstrual fever was highly correlated with HTR1A deficiency. This study aimed to illustrate the molecular mechanisms underlying the clinical observation in this patient. We thoroughly screened HTR1A sequences and identified the -480delA mutation in the promoter region in this patient. We hypothesized that HTR1A -480delA mutation may be responsible for the observed clinical phenotype through the interaction with nuclear factor to impair HTR1A function. Interestingly, dual-luciferase reporter assays also revealed that HTR1A -480delA mutation decreases reporter gene activity. Using biotinylated DNA probe, electrophoretic mobility shift assay (EMSA), and competition assay showed that multiple nuclear proteins specifically bind to this sequence motif. Followed by biotin pull-down assay and LC-MS-MS analysis, we successfully identified poly(ADP-ribose) polymerase (PARP-1) as one of the potential candidates that binds to HTR1A promoter motif. The DNA-protein interaction was further confirmed by chromatin immunoprecipitation (ChIP) assay. Overexpression and knockdown of PARP-1 result in suppressed and enhanced expression of HTR1A, respectively. These data suggested that PARP-1 functions as a repressor of HTR1A expression. Furthermore, HTR1A -480delA mutation causes an increased interaction between PARP-1 and HTR1A, and consequently, results in a further reduction of HTR1A expression. Reduction of expression of HTR1A may thus disrupt HTR1A-mediated hypothermic responses and cause the phenotype we observed in the clinical case. Since HTR1A is an important autoreceptor for serotonin transmission, results from our study will provide valuable information for the role of HTR1A in serotonin-related diseases.

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