以同步定量聚合酶鏈反應偵測精神分裂症病患及其家屬在1q21.1與2q22.1之拷貝數變異

Abstract

摘要 背景與目的：拷貝數變異 (Copy Number Variation) 已經被提出是精神分裂症易感受性的重要原因之一，過去研究指出拷貝數變異在精神分裂症病患上，有著和“常見疾病是由罕見遺傳變異引起”假說並行不悖的特性。因此，用一種有效率且可信賴的方式在精神分裂症的病患上找尋可能和疾病相關的拷貝數變異便成了一個重要的課題。在這個研究中，我們透過精神分裂症病患及其家屬的樣本，希望能評估用兩階段的定量聚合酶鏈反應 (quantitative polymerase chain reaction) 來找尋位在1q21.1以及2q22.1兩段區域的拷貝數變異之實際可行性。 方法：研究樣本是來自臺灣精神分裂症連鎖研究 (Taiwan Schizophrenia Linkage Study)，收集了來自全臺灣手足皆感染精神分裂症的病患及其一等親家屬，共有2462名個案，分別來自607個家庭。利用ABI7900以及TaqManR Copy Number Assays偵測三段區域上拷貝數變異的探針，其中兩個位在2q22.1 (HNMT gene) 一個位在1q21.1 (GJA8 gene)，第一階段基因型定型 (genotyping) 的每一樣本皆以二重複進行，以臨界週期數差異 (△Ct)的中位數 ± 3*標準差作為標準，超出這個範圍的即視為離群值，帶有離群值的個案以及他們的家屬，以四重複的方式進行第二階段的基因型定型，接著透過檢視拷貝數變異在家族中的基因型分佈來判定該拷貝數變異是家族遺傳傳遞或突變而來。至於拷貝數變異與精神分裂症之關係則會利用FBAT軟體進行分析。 結果：根據臨界週期數差異，個案被分成帶有三種不同基因型的組別，分別是：1/0 (deletion, 在該區域上有序列缺失，△Ct > 中位數 + 3*標準差)、1/1 (野生型, 在該區域之序列正常，△Ct 位在中位數 ± 3*標準差間)以及2/1 (duplication, 在該區域上有序列重複出現，△Ct < 中位數 - 3*標準差)。在第一階段的實驗中，在三個區域分別有8名 (CNV marker 1) 、2名 (CNV marker 2) 以及25名 (CNV marker 3) 帶有異常的臨界週期數差異，經第二階段實驗的驗證後，在三個區域分別只有1名 (CNV marker 1)、0名 (CNV marker 2)、13名 (CNV marker 3) 個案的臨界週期數差異仍維持是離群值，這些人被視為帶有確定的拷貝數變異。共計在CNV marker 1有一個家族帶有duplication，而在CNV marker 3有6個家族帶有deletion。FBAT的分析中，顯示deletion的allele帶有會增加罹病危險性的趨勢，但統計上則未達顯著 (z-statistics = 1.134, P = 0.257)。 結論：這個研究展示了一種利用定量聚合酶鏈反應針對特定區域搜尋拷貝數變異並且節省成本的實驗設計，結果指出在我們研究中找到的拷貝數變異大多是經由家族性傳遞。

AABSTRACT Background and Objective: Copy number variations (CNVs) have been postulated to be an important source of genetic susceptibility to schizophrenia. Previous findings on CNVs in schizophrenia patients appear to be compatible with the proposition of “common disease, rare variants.” Under this circumstance, it is important to search for CNVs in individual patients with schizophrenia in an efficient but robust way. In this study, we aimed to evaluate the feasibility of a two-stage quantitative polymerase chain reaction (qPCR) approach in detecting CNVs in two chromosomal regions, 1q21.1 and 2q22.1, in patients with schizophrenia and their families. Methods: The sample consisted of 2462 participants from Taiwan Schizophrenia Linkage Study, which recruited sib-pairs co-affected with schizophrenia and their first-degree relatives throughout Taiwan with a total of 607 families. Three CNV markers were chosen for genotyping using TaqManR Copy Number Assays on the ABI7900, with markers 1 and 2 located on 2q22.1 (HNMT gene) and marker 3 on 1q21.1 (GJA8 gene). First-stage genotyping was performed with 2 replicates of each subject. Then the outliers with a △Ct value beyond median ± 3SD and their family members were re-assayed with 4 replicates. After the second-stage genotyping, we examined the pedigrees of those subjects with confirmed CNV to determine whether the CNV was due to familial transmission or a de novo one. The relations of the CNVs to schizophrenia were evaluated using family-based association analysis. Results: Subjects were categorized to 3 distinct genotypic groups according to their △Ct values: 1/0 (deletion, i.e., > median+3SD), 1/1 (wild type, within the range of median ± 3SD) and 2/1 (duplication, i.e., < median - 3SD). In the first-stage genotyping, there were 8, 2, and 25 subjects counted as outliers for CNV markers 1, 2 and 3, respectively. Among them, 1 subject for marker 1, 0 subject for marker 2, and 13 subjects for marker 3 remained as outliers in the second-stage genotyping, whom were judged to be carriers of confirmed CNVs. In total, there was one family with confirmed duplication at CNV marker 1, and six families with confirmed deletion at CNV marker 3. The deletion at CNV marker 3 tended to be associated with schizophrenia, though not reaching statistical significance in family-based association analysis (z-statistics = 1.134, P = 0.257). Conclusion: This study demonstrated an experimental design that is cost-saving in searching for CNVs using qPCR for targeted regions. The results revealed that most of the CNVs found in families of patients with schizophrenia were due to familial transmission.