近年來，有許多的女性深受乳癌及卵巢癌之苦，而BRCA1和BRCA2是與罹患Hereditary breast-ovarian cancer syndrome（HBOC）有關的兩個重要的基因。這兩個腫瘤抑制基因（tumor suppressor genes）參與在雙股DNA同源重組修復（Homologous recombination repair, HRR）的作用機制中。據研究統計指出，當BRCA1基因在女性體內發生突變時，該名女性將有65-85%的機率罹患卵巢癌，有40-50%的機率罹患乳癌；當BRCA2基因具有突變時，女性則將有40-85%的機率罹患卵巢癌，10-25%的機率罹患乳癌。具有BRCA1 / 2這種生殖系突變（germline mutations）的一般女性，可以透過接受乳房切除術和/或輸卵管卵巢切除術，來預防癌症的發生， 因此正確的診斷對她們來說非常重要。現在已經有許多的方法用於分析BRCA基因的突變，包括Sanger sequencing、解離式高效能液相層析儀（DHPLC）、次世代定序（Next generation sequencing）、mRNA檢測分析、minigene constructs……等等。其中也有很多的BRCA1/2基因遺傳變異型式已經被研究出來，包含點突變（point mutations）、小片段插入/缺失（Small deletion/insertions）、剪接位改變（Splice site alterations）和大片段基因重組 (Large genomic rearrangement, LGR)。有些突變型已被研究指出為致病性的，且在分類級別中被歸類在「Pathogenic」，其他的類別包含「Likely pathogenic」、「Uncertain significance (VUS)」、「Likely benign」及「Benign」共五大類。在這幾個分類級別中VUS 在診斷上是最難被處理的。舉例來說，一個新穎的BRCA變異型被發現位於剪接位置附近，即便它透過生物資訊分析預測為會對RNA剪接造成影響，卻仍被歸類為VUS。以生物資訊分析預測作為唯一證據，採取果斷的臨床行動（如乳房切除術）是非常危險的，而剪接變化的實驗證實是為臨床決策提供更多證據的一種方式。實驗驗證剪接改變（splicing alterations）是提供臨床診斷證據的方法之一，我們實驗室在先前的實驗中，從28個卵巢癌的女性患者中，發現兩個新穎且位於RNA剪接位置附近的VUS，NM_007294.3(BRCA1): c.442-22_442-13del及NM_007294.3(BRCA1): c.4358-8 A>G。在我們的研究中，利用RNA的分析、軟體預測剪接影響和minigene constructs去分析這兩個新穎的VUS案例。我們首先將這兩個案例及兩個已知為「pathogenic」突變型的序列，用生物資訊分析的方式，送到預測剪接（splicing predicrion）影響的軟體網站去預測結果，並比對這些突變位置原本的野生型（wild-type）序列，來做為一個影響RNA剪接的參考結果。然而對於這些預測的網站軟體，一些內含子變異（intronic mutation）是很難判讀其對RNA剪接的影響。所以我們下個步驟就是希望透過minigene constructs的實驗方式來驗證這些BRCA1/2變異對RNA剪接的影響。我們將一個已知影響RNA剪接的變異型作為對照組，和這兩個新穎的突變型分別到pSPL3載體。最後轉染質體DNA到細胞，抽取其RNA來分析突變對RNA剪接的影響。發現NM_007294.3(BRCA1): c.4358-8 A>G突變型對RNA剪接並沒有影響，但NM_007294.3(BRCA1): c.442-22_442-13del透過實驗證實了此突變對RNA剪接造成使mRNA往前多了59個內含子鹼基，並造成移碼（frameshift）轉譯出不正常的BRCA1蛋白，這個結果能加以佐證預測時所得的結果。通過我們建立的實驗系統，將預測RNA剪接變異的分析結合實驗的驗證，能幫助分級發生在內含子上的RNA剪接位附近的VUS變異型，並幫助臨床的實驗分析。

Today, a lot of women have suffered from hereditary breast-ovarian cancer syndrome (HBOC). Women with a mutated BRCA1 or BRCA2 have a higher chance of developing ovarian and breast cancers in their lifetime. Therefore, correct diagnosis is very important for women with germline mutations of BRCA1/2 to receive mastectomy and/or tubo-oophorectomy for cancer prevention. As a result. There are several methods to analyze BRCA mutations and many genetic variations of BRCA1/2 had been reported. Some of the variations are known to be classified in a five-tier classification system. Among them, variant of uncertain significance (VUS) is most troublesome for molecular diagnosis and taking decisive clinical actions. Previously we tested 28 patients with ovarian cancers and found two novel VUS BRCA1 variants near splice sites. In our study, we used splicing prediction in silico and minigene constructs to analyze these two cases. We firstly input the sequences of mutated and wild-type variants into the splicing prediction programs and compared the predictive results. We observed the prediction tools were not so correct to some intronic mutations. Next, we used minigene constructs to investigate the RNA sequences of the mutated and wild-type variants. Taken together, we suggested that it is better to combine predictive models for splicing mutations with experimental results to validate the real splicing effect of the variants under study. The methods established to validate the effect of splicing mutations can help future clinical examinations.

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