利用全基因體定序方法分析臺灣族群脊髓肌肉萎縮症的帶因情形及疾病診斷

Abstract

我們已知東亞族群的脊髓肌肉萎縮症(spinal muscular atrophy)帶因頻率介於1.24%至3.97%之間，其中一篇針對臺灣孕婦進行的大型研究報告，利用多重連接探針擴增技術(multiple ligation-dependent probe amplification)得到了2.10%的帶因率。有鑑於拷貝數變異和SMN1與SMN2基因之間高度同源性所帶來的挑戰，本研究旨在評估全基因體定序(whole genome sequencing)方法在分析臺灣族群脊髓肌肉萎縮症的帶因情形和疾病診斷上的表現。我們分析了臺灣人體生物資料庫(Taiwan Biobank)中1492個全基因體定序資料，使用Illumina DRAGEN SMN Caller來辨識SMN1和SMN2的拷貝數變異(copy number variation)，並用多重連接探針擴增技術進行驗證。我們也使用Illumina DRAGEN Small Variant Caller來檢測單核苷酸變異(single nucleotide variant)或插入缺失變異(insertion and deletion)，並通過整合基因組數據之視覺化工具(Integrative Genomics Viewer)確認變異的位點。最後我們找到了23個SMN1拷貝數缺失的帶因者，帶因率為1.56%，並確認SMN Caller的分析結果與多重連接探針擴增技術所得結果全數吻合。此外，我們透過SMN Caller和其他策略分析了兩例脊髓肌肉萎縮症患者的全基因體定序資料，確認皆帶有SMN1基因上不同變異的複合異合子(compound heterozygous)。全基因體定序技術可以用來篩檢脊髓肌肉萎縮症的拷貝數變異，若能整合其他生物資訊分析工具，將能突顯定序的優勢，並運用於未來的擴大型帶因篩檢和新生兒篩檢當中。

Background The carrier frequency of spinal muscular atrophy (SMA) in the East Asian population has been reported to range from 1.24% to 3.97%. Using the multiple ligation-dependent probe amplification (MLPA) method, the most extensive study on Taiwanese pregnant women found a carrier frequency of 2.10%. Even though there are issues of structural variation and high sequence similarity between SMN1 and SMN2 genes, several studies have strived to develop sequencing-based solutions over the past decade. This study aims to assess the performance of whole genome sequencing (WGS) technology and computational tools in estimating carrier status and diagnosing patients in Taiwan. Methods 1492 subjects with whole genome sequencing data from Taiwan Biobank were screened to identify spinal muscular atrophy mutation carriers. SMN1 and SMN2 copy numbers were determined using Illumina DRAGEN SMN Caller and validated by multiple ligation-dependent probe amplification. In addition, we applied the same pipeline and Illumina's DRAGEN Small Variant Caller to detect copy number variation and other variants in two spinal muscular atrophy patients, which were verified by Integrative Genomics Viewers (IGV) and further validated with bioinformatic algorithms and orthogonal methods. Results Among 1480 Taiwan Biobank samples, 23 subjects with one SMN1 gene copy were identified as spinal muscular atrophy carriers, resulting in a carrier frequency of 1.56%. Multiple ligation-dependent probe amplification confirmed the results of these 23 carriers and 10 normal participants with various SMN1 and SMN2 copy number combinations. Furthermore, both spinal muscular atrophy patients were confirmed to have compound heterozygous variants with one copy number loss of the SMN1 gene and small variants located on the other SMN1 allele. Conclusion Using whole genome sequencing for spinal muscular atrophy carrier screening is feasible and promising. By integrating additional bioinformatics analytical tools, we can better leverage sequencing technology's strengths, facilitating its implementation in future expanded carrier screening and newborn screening.