應用生物晶片及唐氏症羊水細胞探討第三條21號染色體對基因表現變異性之影響

研究目的唐氏症是目前造成智能不足的最大原因，但至今我們對唐氏症的瞭解，僅止於多一條第21號染色體，至於多一條第21號染色體如何造成各種不同的唐氏症症狀，仍然不甚明瞭。此外，唐氏症屬於一個多症狀的症候群，其表現並不一致而呈現相當大的變異性(variation)，在我們進一步想要瞭解唐氏症的病態生理學前，必須要先掌握唐氏症症狀的變異性。然而目前有關唐氏症症狀和基因表現變異性的研究，付之闕如，因此我們研究的目標，就是測量唐氏症組織的基因表現變異性(variation)，和正常組織比較起來是否有所不同，並進一步想要解釋，為何唐氏症病人的表現如此多樣性。研究方法第一部份我們應用生物晶片(microarray)的技術、及使用可當成比較基準的標準化參考RNA（reference RNA），比較唐氏兒(三染色體21症)之羊水細胞，以及正常羊水細胞的基因表現，觀察在唐氏症羊水細胞中，是否有變異性增加的現象，並以chi-square test檢定不同變異數(variance)組群的基因數目差異。我們使用6個正常羊水細胞檢體及5個唐氏症羊水細胞檢體，完成RNA抽取、RNA品質分析、晶片結合、以及數值正常化(normalization)等步驟。訊號經過讀取之後，分別計算出唐氏症羊水及正常羊水間，所有基因的變異數(variance)，再根據變異數予以分組，並統計不同組別之內的基因個數。此外我們進一步篩選在正常細胞中變異性不大的基因，卻在唐氏兒羊水細胞內出現變異量變大的基因，其變異性(variance)的差異，以F test檢定之。並進一步使用相關技術如QRT-PCR，確認這些在正常細胞表現正常，卻在唐氏症細胞中表現失去一致的基因，其變異性增加現象的確存在於各羊水細胞，而其變異性增加現象，也有可能是導致唐氏兒多變化症狀的原因。最後，我們模擬上述實驗的條件並建立亂數模型，模擬新的一批唐氏症羊水細胞檢體及正常羊水細胞上做分析比較，看是否具有相同的效力，以驗證我們的結果並非來自偶然。當然在整個過程中，嚴格控制實驗誤差是相當重要的。第二部分我們下載國外已發表的唐氏症胎兒腦部生物晶片的資料，並用同樣的方法運算，觀察其是否有相同的變異性增加的現象，以驗證變異性增加的現象不只存在於羊水細胞，也同樣發生在其他組織。結果以整個細胞基因表現而言，我們發現在唐氏症羊水細胞檢體，第21號染色體上的基因有表現增加的趨勢，其平均表現量約為正常細胞的1.35倍，這個發現跟之前的研究結果相似。我們在唐氏症羊水細胞檢體內可以發現變異數大於1的基因數目和正常羊水細胞檢體比較起來明顯增加。在所有21003個基因中，我們用F test檢定篩選出37個基因，其變異數在唐氏症和正常羊水細胞檢體中有明顯不同，並經過pFDR校正，其中pFDR value<α=0.05為有意義。其中過濾掉前景和背景差異小於2倍，且其變異性在唐氏症羊水細胞檢體中明顯增加者共15個基因。其中我們挑出可能在生物發育扮演重要角色的四個基因(CXCL12, GABRA5, ATP5G3 and ZZZ3)，並增加檢體數目為八對唐氏症羊水細胞檢體及正常羊水細胞檢體，進一步做QRT-PCR的驗證，發現其中CXCL12出現有意義的變異性差別。此外，我們所建立的亂數模型，所挑選出來的基因和實驗所挑出來的基因有明顯的差異。在第二部分實驗，我們下載唐氏症胎兒腦部資料，並加以計算，發現變異數增加的基因數目，其增加的趨勢更為明顯。結論在唐氏症組織中，不論是羊水細胞檢體或是大腦細胞檢體，在多一條21號染色體的情況之下，會使某些基因的表現變異性增加，這種增加並非來自偶然，並且可以用QRT-PCR的方法予以驗證。這些基因表現的變異性增加也許是某些唐氏症症狀多變化的主因，但究竟是哪些基因造成哪種特定的性狀，使其基因表現的變異性直接影響性狀表現的變異性，則需要進一步動物實驗去驗證。

關鍵字

唐氏症；基因變異性；生物晶片；羊水細胞

並列摘要

Introduction: Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. With more publications focusing on over-expression on genes of chromosome 21 and expression variation in normal human and organisms, the research for significance of gene expression variation in DS is lacking. We use microarray to study the effect of extra chromosome 21 to gene expression variation in amniocytes of trisomy 21. We also observe if this change of variation occurred in fetal brain tissues that the microarray datasets were shared on the websites. We hoped to get a new insight of genomic biology in trisomy 21. Method and materials: We use 5 amniocyte cultures of trisomy 21 and 6 cases of normal amniocytes. Total RNA was extracted, mixed with human reference RNA and hybridized with microarray chips (Agilent Technologies, USA). Normalization used LOWESS method. The variances of 21073 genes of 2 groups was calculated and graded into different groups. The gene numbers of different variance was compared between the DS and normal control. And χ2 method was used to test the significance. The expression of genes that vary among amniocytes of trisomy 21 and normal control was evaluated using ANOVA model and p-value was modified with the concept of pFDR. Further validation of 4 genes (CXCL12, GABRA5, ATP5G3 and ZZZ3) with significantly difference in variance was performed by quantitative RT-PCR. The same statistical methods were also applied on the dataset of Mao’s fetal trisomy 21 brain tissues on the websites. To determine how many genes are expected to be in common by chance if genes were chosen randomly, we undertook two simulation studies with 2000 datasets generated by randomly using and the same analysis for 100 datasets in our study. Results: Average expression ratio of Down syndrome over normal control in genes on chromosome 21 was 1.35. And other genes of non-chromosome 21 keep the ratio at 1. The numbers of genes with variance more than 1 is much increased in cells of trisomy 21 in our amniocytes dataset and Mao’s fetal brain dataset(p<0.01). When performing the F-test, 37 hypothesizes will be rejected. And 15 genes will be picked up after excluding weak signals. The difference of variance does not come from random. CXCL12 was validated to have different variance in two groups by QRT-PCR(p=0.003). Conclusion: In human trisomy 21 tissues including amniocytes and fetal brain tissues, the gene expression variation increased generally as compared to normal tissues. There exist a set of genes with increased variance in gene expression and can be validated by QRT-PCR. Increased variation of gene expression in trisomy 21 may lead to variable level of mRNA and then variable phenotypes of Down syndrome.

並列關鍵字

Down syndrome ； gene expression variation ； microarray ； amniocyte

參考文獻

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Antonarakis SE, Epstein CJ. The challenge of Down syndrome. Trends Mol Med. 2006, 12: 473-9

Busciglio J, Pelsman A, Wong C, Pigino G, Yuan M, Mori H, Yankner BA. Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down's syndrome. Neuron. 2002, 33: 677-88

Cheon MS, Bajo M, Kim SH, Claudio JO, Stewart AK, Patterson D, Kruger WD, Kondoh H, Lubec G. Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II). Amino Acids. 2003a, 24: 119-25

Cheon MS, Kim SH, Ovod V, Kopitar Jerala N, Morgan JI, Hatefi Y, Ijuin T, Takenawa T, Lubec G. Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part III). Amino Acids. 2003b, 24: 127-34

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應用生物晶片及唐氏症羊水細胞探討第三條21號染色體對基因表現變異性之影響

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