AlphaThaICNV: 通過次世代定序技術增強 α地中海型貧血拷貝數變異檢測能力

Abstract

引言 α-地中海貧血是一種常見的血紅蛋白病，由α-珠蛋白基因（HBA）的缺失引起，導致α-珠蛋白的生產減少或完全缺乏。這會導致小紅細胞增多症、貧血，以及在嚴重情況下引發如肝腫大和需依賴輸血等併發症。鑒於其對全球健康的負擔，準確、高效且具規模化的分子診斷方法對於α-地中海貧血攜帶者的篩查與診斷至關重要。傳統方法如單管多重PCR僅限於檢測已知的缺失，而全外顯子組測序（Whole-Exome Sequencing）因缺乏靈敏度，難以準確檢測幾乎相同的HBA1和HBA2基因中的拷貝數變異（CNVs）。

材料與方法 AlphaThalCNV流程利用GATK-gCNV工具，並通過優化參數（區塊大小：50；填充：400），提升HBA基因簇內的CNV檢測能力。該流程以100個隨機樣本的群體訓練模型，實現群體內的CNV檢測。隨後，對每個α-地中海貧血病例進行單獨分析，並將結果與建立的模型比較。結果使用IGV可視化並導出為VCF文件，根據被刪除或重複的α-珠蛋白基因數量進行分類。

為驗證GATK-gCNV結果的準確性，我們採用了Samuel S. Chong等人改編的引物進行多重PCR，可在單次實驗中檢測多個基因缺失。群體分析納入了415名來自台灣的個體，統計分析α-地中海貧血攜帶者的盛行率。

結果 AlphaThalCNV成功檢測到三個此前未檢出的α-地中海貧血病例，包括兩個--SEA缺失和一個-3.7缺失。此外，五名最初因其他疾病住院的α-地中海貧血攜帶者也被準確檢測並通過多重PCR確認為真正陽性。

在台灣群體中，AlphaThalCNV檢測到--SEA型兩拷貝缺失的攜帶者盛行率為5.3%，HBA2基因中的一拷貝缺失（-4.2型或-3.7型I/II）的盛行率為2.2%。這些發現與使用其他方法（Hsu等，2023年）進行的大規模研究結果一致，證實了該流程的穩健性與準確性。

結論 AlphaThalCNV流程顯著提升了α-地中海貧血CNV檢測的準確性，特別是在標準流程遺漏的病例中。通過整合精確的分類與可視化工具，該流程為大規模群體分析提供了一種可靠的方法。其適應性使其成為未來醫學診斷和流行病學研究中的重要工具。

Introduction Alpha-thalassemia is a prevalent hemoglobinopathy caused by deletions in the α-globin genes (HBA), resulting in reduced or absent α-globin production. This leads to microcytosis, anemia, and in severe cases, complications like hepatomegaly and blood transfusion dependency. Given its global health burden, an accurate, efficient, and scalable molecular diagnostic approach is critical for α-thalassemia carrier screening and diagnosis. Traditional methods like Single-Tube Multiplex PCR are limited to detecting known deletions, while Whole-Exome Sequencing lacks sensitivity to accurately detect copy number variations (CNVs) in the nearly identical HBA1 and HBA2 genes.

Materials and Methods The AlphaThalCNV pipeline leverages the GATK-gCNV tool with optimized parameters (bin size: 50, padding: 400) to enhance CNV detection in the HBA gene cluster. The pipeline was trained using a cohort of 100 random samples, enabling CNV calling within the cohort. Subsequently, α-thalassemia cases were analyzed individually against the constructed model. Results were visualized with IGV and exported as VCF files for further classification based on the number of alpha-globin genes deleted or duplicated. To confirm the accuracy of GATK-gCNV results, we employed Multiplex PCR with primers adapted from Samuel S. Chong et al., enabling detection of multiple deletions in a single run. The cohort analysis included 415 individuals from Taiwan to statistically analyze α-thalassemia carrier prevalence.

Results AlphaThalCNV successfully identified CNVs in three previously undetected α-thalassemia cases: two with --SEA deletions and one with -3.7 deletion. Additionally, five carriers of α-thalassemia, initially hospitalized for other diseases, were accurately detected and confirmed as true-positive through Multiplex PCR. Among the Taiwanese cohort, AlphaThalCNV revealed a carrier prevalence of 5.3% for two-copy deletions (--SEA type) and 2.2% for one-copy deletions in the HBA2 gene (-4.2 type or -3.7 type I/II). These findings align with larger-scale studies conducted via alternate methodologies (Hsu et al., 2023), demonstrating the pipeline's robustness and accuracy.

Conclusion The AlphaThalCNV pipeline has significantly improved the accuracy of α-thalassemia CNV detection, particularly in cases missed by standard pipelines. By integrating precise classification and visualization tools, the pipeline offers a reliable method for large-scale cohort analysis. Its adaptability makes it a valuable tool for future medical diagnostics and epidemiological studies.