改進RYR1基因變異點之功能性分析以評估對惡性高熱症之致病性

Abstract

惡性高熱 (Malignant hyperthermia) 是一種與骨骼肌相關的藥理遺傳學疾病，在臨床上具重要意義。會對強效揮發性麻醉氣體，如七氟醚 (sevoflurane) 或琥珀膽鹼 (Succinylcholine) 等，產生高代謝反應。其病程快速猛烈，若沒有及時給予唯一的治療藥物單挫林 (Dantrolene)，可能引起橫紋肌溶解或多重器官衰竭而死亡。 會引起惡性高熱的基因主要為RYR1基因，與此基因相關的病例占了惡性高熱總病例的50%-70%。現今因次世代定序 (Next-generation sequencing) 的發展，使我們能建立出快速並精準的惡性高熱相關基因檢測，透過此基因檢測我們找出許多以往還未發現的變異點，尤其此為針對國人的常見變異點，但這些變異點的致病性還無法被準確辨認。 現行國際的惡性高熱指南，對變異點致病性的確認相當嚴謹，目前已發現超過400種變異點，但只有其中的48個被證實其具有致病性。若我們為疑似有惡性高熱的病人進行基因檢測時，只要發現的變異點不在此內，我們就無法確定此變異點與惡性高熱之臨床相關性。因此，我們需要快速發展一個既可靠又具經濟效益的RYR1功能性分析，以確認各個變異點之致病性。 先前主要的功能性研究是使用兔RYR1的cDNA來進行，而非人類的RYR1。後續重要的細胞試驗也以HEK293細胞來進行，而非人類的骨骼肌細胞；以科學的角度來說，我們並不那麼滿意。因此，我們希望能發展一種功能性分析，是以人類的RYR1 cDNA為基礎，並在人類骨骼肌細胞中進行試驗，以此實驗架構來測試我們預計要了解其功能性的變異點。 因此，我們設計一組含有人類RYR1 cDNA的質體，並加入IRES (internal ribosome entry site)、組蛋白 (histone) H2B、紅色螢光蛋白mCherry、和轉位子 (transposon) PiggyBac，以穩定基因表現，同時方便實驗分析。 我們利用綠色螢光蛋白eGFP (以代替RYR1基因) 和HEK293細胞來先測試我們建構的質體表現，經由螢光顯微鏡下可觀察到，其在轉染作用 (transfection) 下還是能夠表達。在流式細胞儀的分析上也可以很明顯地看到HEK293細胞經過目標質體的轉染作用後存活率並未因此下降，在轉染作用的效率上也有33.2%。 同時，我們使用定點突變 (Site-directed mutagenesis PCR) 的方式，對先前研究找出的，望能確認其致病性之五個變異點 (c.487C>T、c.1565A>G、c.2677G>A、c.6488G>T和c.7042_7044del)，針對上述變異點之形式，在我們原來的質體上做變更，以利後續執行功能性研究。 最後，我們收入兩則與惡性高熱相關之臨床病例，來演示在臨床中遇到惡性高熱患者時，會產生的遺傳諮詢，與在實務上的運用。

Malignant hyperthermia is a pharmacogenetic disease of clinical importance associated with skeletal muscle. It produces a hypermetabolic response to potent volatile anesthetic gases such as sevoflurane or succinylcholine. The course of the disease is rapid and violent, and death may result from rhabdomyolysis or multiple organ failure if Dantrolene, the only therapeutic agent, is not administered in a timely manner. The gene that causes malignant hyperthermia is mainly the RYR1 gene, which accounts for 50%-70% of all cases of malignant hyperthermia. Nowadays, the development of next-generation sequencing has enabled us to establish a rapid and accurate test for related malignant hyperthermia genes, through which we can identify many previously undetected variants, especially those common variants in Taiwanese population, but the pathogenicity of these variants cannot be accurately identified. The current international guidelines for malignant fever are very stringent in confirming the pathogenicity of variants, and over 400 variants have been identified, but only 48 of them have been confirmed to be pathogenic. If we perform genetic testing for patients suspected of having malignant hyperthermia, we will not be able to confirm the clinical relevance of the variant as long as it is not included here, which is a major problem for patients. Therefore, we need to rapidly develop a reliable and cost-effective functional analysis for RYR1 to confirm the pathogenicity of each variant. Previously, the main functional studies were performed using rabbit RYR1 cDNA rather than human RYR1 cDNA, and subsequent important cellular experiments were performed with HEK293 cells rather than human skeletal muscle cells, which is not as scientifically satisfactory as we would like. Therefore, we would like to develop a functional assay based on human RYR1 cDNA and perform experiments in human skeletal muscle cells as an experimental framework to test the variants that we expect to understand their expression. Therefore, we designed a set of plasmids containing human RYR1 cDNA and added IRES (internal ribosome entry site), histone H2B, red fluorescent protein mCherry, and PiggyBac transposon to stabilize gene expression and facilitate experimental analysis. We used eGFP, a green fluorescent protein (instead of RYR1 gene), and HEK293 cells to test the performance of our constructs. It is also clear from the flow cytometry analysis that the survival rate of HEK293 cells did not decrease after transfection with the target plasmid, and the efficiency of transfection was 33.2%. At the same time, we used site-directed mutagenesis PCR on the original plastid to modify five mutation variants (c.487C>T, c.1565A>G, c. 2677G>A, c.6488G>T and c.7042_7044del) were changed to facilitate subsequent functional studies. Finally, we present two clinical cases related to malignant hyperthermia to provide an in-depth understanding of the genetic counseling and practical application in clinical situations where patients with malignant hyperthermia are encountered.