利用微陣列和FISH分析唐氏症患者基因的表現和3D立體空間表現

Abstract

唐氏症(Down syndrome,DS)為人類引起智障，和非整倍體數目異常(aneupliody)最常見的染色體異常。就我們所知唐氏症的決定區域是在第二十一號染色體的長臂上一段小小的區域，即21q22.1∼21q22.3；只要這一區域有三套，臨床上就會看到唐氏症的表現型(phenotype)。但目前引起唐氏症，其基因劑量的表現尚不清楚。依文獻得知所有的基因都存在於染色體上，基因的功能受到其在染色體上之位置與環境的調控。要瞭解基因的功能有必要瞭解基因和染色體在細胞核內的空間結構。在細胞週期內，gene activity是在interphase時而非在metaphase時，因此我們必需探討在interphase時，細胞核內的染色體到底是以什麼樣的狀態存在呢？同時由於目前微陣列(Microarray) 是發展及應用較為成熟的生物晶片技術之一，此晶片技術已廣泛的應用於生物醫學領域，如癌症、藥理、毒理學、感染、細胞分化、發育及生殖醫學等，亦將直接或間接的應用於疾病之診斷、分類、預後評估，並改善疾病之治療。 於是本論文則經由FISH(Fluorescence in situ hybridization)和最新生物晶片微陣列(Microarray)，來分析DS interphase之3D空間分布和基因的表達。我們應用two-sample t-test, Welch’s test 或 Significance analysis of microarrays (SMA)來分析microarray基因的表達。然而FISH則應用Mathcad 軟體分析唐氏症和正常人細胞，包括羊水和血液細胞之interphase。 結果顯示在調查22,572的基因探針時，發現有29個基因是有意義。而這29個基因中有18個基因(63%)位於21號染色體(HAS21)上，而這18個基因主要與基因調節細胞間的溝通(genes regulating cellular communication)，新陳代謝(metabolism) 和分化(differentiation)有關。有關DS在interphase時之3D立體結構，本研究發現21號染色體和18號染色體的空間相對位置，有一定的比值在0.8~1.2的範圍，而且男女沒有差異。而對照組21號染色體和X號染色體的空間相對位置，也能找出其比值，並且不同於21號染色體和18號染色體空間之比值。所以染色體在interphase時，其在空間的territory可能是固定而非random。同時結果證實，三點紅色(21號染色體)中有二點是非常近，是有意義P<0.05。而且最短的c，並沒有落在比值在0.8~1.2的範圍，所`以知道最短的二點21，其中一點為多出來的另一個21號染色體。只是知道最靠近二點21之一為異常21號染色，至於是那一點必須近一步實驗證實。本研究目前已經知道，唐氏症一些有用的基因和3D空間概念，希望進一步能應用在疾病預防、預後評估，並改善疾病之治療。

The gene expression of Down syndrome (DS) is the most dubitative culprit for the etiology of mental retardation and aneupliody in human. The extra copy of the proximal part of chromosome 21q22.1~21q22.3 appears to result in the phenotypic DS: mental retardation, characteristic facial features, hand anomalies, and congenital heart defects. However, the gene profile of chromosome 21 in DS is not clear. In this project, we use FISH (Fluorescence in situ hybridization ) and Microarray techniques, to interpret the spatiotemporal contous of gene expression on the DS chromosome 21 at the interphase of mitosis . To analyze the patterns of gene expression from microarray, we apply the scoring systems including two-sample t- test, Welch’s test or Significance analysis of microarrays (SAM) test in the two-group comparative experiments according to the principle of data fusion in information retrieval to combine the multiple scoring systems. A rank/score function is used as a predictive variable for the effectiveness of combination to improve the identification of significant genes. In addition, we use Mathcad software system to analyze results from FISH performed on the DS cells/normal cells from amniotic fluid / blood. The results from microarray showed that: 1. there is no difference in gene expression in term of gender whether the gene is derived from DS patient or healthy individual. 2. Out of 22572 genes, 29 were detected significantly different between DS and healthy people. Furthermore, 18 out of these 29 positive genes were found on the chromosome 21, and are thought to perform the functions of cellular communication, metabolism, and differentiation. Using FISH to stain the chromosomes at interphase, we found that the spatiotemporal territory of each pair of gene on chromosome belonging to healthy people is not random disregarding which type of cells examined. Furthermore, we found that there is an extra spatiotemporal territory of genes on the DS chromosome 21. The length of this surplus territory is rather short and out of the normal range. In conclusion, we think that the 18 different genes found on the DS chromosome 21 are located on the surplus short piece of territory , and these extra genes may result in the dosage effects which drive the functions of cellular communication, metabolism, and differentiation aberrant.