以次世代定序檢測兒童嗜血症候群病患之基因變異

Abstract

論述重點 本研究目的為使用全外顯子定序，分析台灣兒童嗜血症候群相關基因變異之分布情形、與臨床表現及預後之關係。嗜血症候群是一類罕見的症候群，症狀以發燒、全血球低下、多重器官衰竭為主，進展通常十分快速，若無法早期診斷加以即時的治療，常危及生命。雖然嗜血症候群病患的表現類似，但其成因複雜且非單一機轉可以概括。先前已有許多研究發表可在符合HLH-2004診斷標準的兒童嗜血症候群病患檢測出潛在性致病之基因變異。另一研究將全外顯子定序整合到嗜血症候群的診斷流程中，指出此檢測方式可在許多先前已接受針對家族性嗜血症候群基因群定序篩檢後無特殊發現的病患中，發現其他的致病機轉；當中包含一些可受惠於特定治療方案如造血幹細胞移植或標靶治療的疾病，如可造成先天性免疫缺乏及自體發炎的單基因疾病。由此可見全外顯子定序檢測有其附加價值。

方法 本研究的收案對象為臨床診斷為嗜血症候群之兒童，包含符合HLH-2004診斷標準的病患，以及部分符合此標準但合併血球吞噬現象的病患。取得符合收案標準之研究對象的周邊血或骨髓檢體後，進一步提取檢體中的基因體DNA進行全外顯子定序分析。初步得到之基因變異，做篩選利用的標準包括：虛擬基因檢測組套、最小等位基因頻率、表型共分離現象、電腦模擬預測、免疫細胞表達程度、與已知疾病相關性、蛋白功能和孟德爾遺傳模式等。經篩選所得之基因變異根據ACMG指引進行分類，並將致病性、可能致病性和不確定意義的基因變異列為潛在疾病致因變異。最後透過Integrative Genomics Viewer、Varsome和Sanger測序進行驗證，並將得到的基因型資訊與臨床數據進行相關分析。

結果與討論 本研究共有57位患者參與，收案區間橫跨25年。透過全外顯子定序，可在台灣47％的台灣兒童嗜血症候群病患檢出可能的致病性變異。在本研究中，大多數測得的變異位於與先天性免疫缺陷相關的基因中，其中可導致控制病原體能力受損的基因變異佔30％；可導致自體發炎的基因變異佔17％。只有5%的受試者在與淋巴細胞毒殺功能缺失相關的基因中帶有可能的致病性變異，其中觀察到一個在不同病患身上重複出現、位於UNC13D之內含子的病理性變異。本研究發現最常見的嗜血症候群誘因為感染和自體免疫疾病；被這兩種誘因之其一誘發的患者中，有一半可測得潛在的分子診斷。整體存活率為43％（95％信賴區間：23％〜61％；追蹤時間之中位數：8.4個月）。在不同嗜血症候群基因型類別之間，並未觀察到整體存活率有顯著差異。

結論 台灣兒童嗜血症候群病患的基因頻譜有其獨特性及多樣性，其中可能的致病性基因變異大多位於和先天性免疫缺乏或自體發炎疾病相關的基因中。作為一個能找出嗜血症候群之潛在基因診斷、得以針對特定疾病作出適當處置之工具，全外顯子定序有其適切性；然而其對於預後的影響，仍需前瞻性研究進一步評估。

Background Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome characterized by clinical features such as fever, pancytopenia, and multi-organ failure. The progression of the disease is rapid, and delayed diagnosis and treatment can have fatal consequences. However, the underlying mechanisms of the disease are diverse. Previous studies have indicated that potentially disease-causing variants were identified in a significant proportion of pediatric HLH patients who met the HLH-2004 criteria. One study integrated whole exome sequencing (WES) into the diagnostic process and suggested that additional pathogenic mechanisms may be uncovered in patients who did not have a genetic explanation based on previous targeted sequencing tests for familial HLH genes. Consequently, specific options like hematopoietic stem cell transplantation or targeted therapy could be considered for patients with monogenic primary immunodeficiency and monogenic autoinflammatory disease. This study aims to explore the genetic landscape of Taiwanese pediatric HLH patients through WES, establish genotype-phenotype correlations, and discover novel variants and genes associated with HLH.

Methods WES was conducted on pediatric patients diagnosed with HLH, either meeting the HLH-2004 criteria or presenting hemophagocytosis with partially fulfilled criteria. Genomic DNA was extracted from peripheral blood or bone marrow cells for the WES analysis. Variant filtering criteria included the use of a virtual gene panel, minor allele frequency cutoff, phenotype cosegregation, in silico prediction, immune cell expression, relevance to known diseases, protein function, and Mendelian inheritance. Selected variants were classified according to ACMG guidelines, with pathogenic variants, likely pathogenic variants, and variants of uncertain significance reported as potential disease-causing variants. Validation was achieved through Integrative Genomics Viewer, Varsome, and Sanger sequencing. The genetic information was then correlated with clinical data.

Results A total of 57 patients were enrolled in this study, spanning a period of 25 years. Through the implementation of WES testing, potential disease-causing variants were detected in 47% of Taiwanese pediatric HLH patients. Within this study, the majority of the identified variants were found in genes associated with inborn errors of immunity, resulting in impaired pathogen control (30%) or autoinflammation (17%). Only 5% of participants had likely disease-causing variants in genes related to impaired lymphocyte cytotoxicity, with the observation of a recurrent intronic UNC13D pathogenic variant. Infections and autoimmune diseases were the most common triggers for HLH, with half of these patients showing an underlying molecular diagnosis. The overall survival (OS) was 43% (95% CI: 23%~61%; median follow-up time: 8.4 months). No significant difference in OS was noted among different HLH genetic profiles.

Conclusion The genetic landscape of HLH phenotype in pediatric patients in Taiwan is diverse, with the majority of potential disease-causing variants identified in genes associated with primary immunodeficiency and autoinflammatory diseases. Notably, only a few variants related to impaired lymphocyte cytotoxicity were identified. This distinct distribution of variants compared to other studies with cohorts of different ethnicities suggests the effect of population stratification. WES proved to be a viable diagnostic approach for uncovering underlying genetic causes of HLH syndrome, enabling disease-specific management strategies. Prospective trials are needed to evaluate the impact of WES on prognosis.