利用斑馬魚胚行為分析評估病患來源RYR1基因突變之致病性

Abstract

RYR1 (Ryanodine receptor type 1)的基因序列在脊椎動物間共享高度的保守性，同時作為目前已知骨骼肌中最大的離子通道蛋白，負責將肌漿網儲存的鈣離子(Ca2+)，在運動時傳導到肌肉細胞質以供肌凝蛋白結合使用。RYR1 基因突變會導致先天性肌病如中央空軸肌病、多重微核心病和肌纖維型不均勻症等亞型發生。本研究根據一起家族性臨床案例設計，家族成員中，患病家屬對於需運用近端肌的日常生活皆表現明顯障礙，子女亦出現相似病徵。肌肉磁振造影在相關肌群也都出現顯著的脂肪浸潤。肌肉病理切片發現部分第一型肌纖維中有明確界線的中央管狀結構，呈現典型中央空軸肌病之病理特徵。全外顯子定序發現病患RYR1 基因上有異形合子錯義突變19q 13.2; NM_000540.2:c.14432C>T，然而目前仍無法判定此突變是否具致病性。 本研究欲利用斑馬魚來建立動物模型並驗證該突變的致病性。相較於禽類或哺乳類，斑馬魚因其體外受精與胚體透明等特性，適合作為發育遺傳研究的模式生物。前人的研究已經證實，斑馬魚(Danio rerio)的ryr1 又表現出兩種同源基因分型，其中慢縮肌主要負責姿態維持，大多表現Ryr1a (Ryanodine receptor 1a,Ryr1a)，而快縮肌大多參與爆發性的游泳行為，主要表現Ryr1b。若斑馬魚ryr1b基因突變，二日齡後可觀察到Ryr1b 功能失常，並導致肌漿網內Ca2+釋放受阻，使肌肉無法正常收縮，進而影響運動功能。而本研究使用CRISPRi 技術，針對ryr1b 基因轉譯起始位點設計gRNA 進行敲低(knockdown)，以模擬蛋白質稀缺狀態，並結合肌肉功能評估與鈣離子濃度量測，建立對應疾病的分子機制模型。隨後透過補償試驗(rescue assay)，檢視功能是否得以改善或恢復以評估病患單核甘酸序列變化的致病性。其中肌肉功能試驗分為游泳行為與逃脫反應兩項試驗。 逃脫反應試驗以塑膠探棒刺激吻部，並透過攝影記錄逃脫反應時間作為評估依據，結果顯示敲低組逃脫所需秒數顯著高於對照組平均所需的200ms，補償RYR1-14429-SNV後，隨著劑量上升，逃脫分數也顯著提升，顯示RYR1-14429-SNV對於骨骼肌收縮具有出現收縮無力的致病性，並且具有明顯的劑量效應。同時將逃脫試驗與qPCR 結果作相關性分析，發現隨著各組間的ryr1b表達量下降，其對應的逃脫時間也顯著上升，兩者間相關係數更為高達0.9808 的負相關。接下來，為了近一步驗證突變位點對於斑馬魚幼魚運動功能的影響，透過建立的游泳行為試驗中，將以光敏化合物Optovin於紫外光照射幼魚誘發瞬時游泳行為，以觀察敲低ryr1b以及補償RYR1-14429-SNV mRNA 對於縱向游泳角度與擺動頻率的影響，其中結果顯示，對照組普遍擺動角度平均介於±20°左右，而敲低組則顯著降低到平均約±8°，同時在頻率以及耐力上也顯著低於對照組，而在RYR1-14429-SNV補償組中，游泳頻率、尾鰭擺動角度以及總游泳時長也都顯著低於對照組及RYR1 補償組，結果更趨近於敲低組，證實突變位點對於游泳行為的致病性，結果中的誘發游泳行為的延遲，也暗示鈣離子傳導能力受影響。為了更微觀定量鈣離子的傳導情況，在鈣離子定量實驗中，可發現RYR1-14429-SNV補償組鈣離子都蓄積於肌漿網內，紅螢光強度顯著高於對照組，相反的，位於細胞質內游離的鈣離子綠螢光訊號，則顯著低於對照組，同時透過紅/綠螢光比值的統計分析判斷出肌漿網與細胞質的鈣離子動態分布情況，也會隨著補償劑量提升而使流動率下降，這結果也解釋了RYR1-14429-SNV 補償組在逃脫實驗骨骼肌收縮顯著無力，是源於突變位點造成RYR1 離子通道傳導能力下降，導致細胞質內可供肌旋蛋白接合的總Ca2+濃度不足的可能性。也因為RYR1-14429 C>T 突變造成RYR1 鈣離子流通率下降，光趨活游泳行為中，才會在經由Optovin 瞬時活化運動行為時，導致游泳最大擺角顯著降低，總游泳趨活時長顯著下降，甚至出現明顯的游泳延遲。上述結果一再證實RYR1-14429 C>T 的致病性，並且透過鈣離子定量結果顯示治病模式更傾向於功能喪失型突變，導致鈣離子傳導能力下降，導致鈣離子蓄積在肌漿網內而無法有效傳導進細胞質供骨骼肌收縮使用，這也或許揭示臨床案例中，近端肌無力家族導致大腿肌無力而影響日常起居的致病機制。 而利用本實驗成功驗證並建立的 ryr1b 敲低-補償模型，期望後續得以作為致病性驗證的模型，釐清病患點突變之致病模式更傾向G-O-F 中RYR1 閉合不全或L-O-F 無法正常傳導Ca2+的蛋白功能型態，也期望能作為人類臨床病患特定RYR1-RM 致病機制驗證平台的基礎。此一策略，不僅有助於精準醫療的推展，也契合實驗動物3R 原則，未來亦可望能推進治療策略的開發與驗證。

The RYR1 (Ryanodine receptor type 1) gene is highly conserved among vertebrates and encodes the largest known ion channel in skeletal muscle. RYR1 mediates the release of calcium ions (Ca²⁺) from the sarcoplasmic reticulum (SR) into the cytoplasm during excitation–contraction coupling, thereby providing Ca²⁺ for actin–myosin interaction and skeletal muscle contraction. Mutations in RYR1 are associated with a spectrum of congenital myopathies, including central core disease (CCD), multiminicore disease, and congenital fiber-type disproportion. In the present study, we investigated a familial clinical case in which the affected patients exhibited pronounced proximal muscle weakness that interfered with daily activities, while their offspring presented with similar clinical manifestations. Muscle magnetic resonance imaging revealed extensive fatty infiltration in the affected muscle groups, and muscle biopsy demonstrated well-defined central core structures in a subset of type I muscle fibers, consistent with the pathological characteristics of CCD. In the results of Whole-exome sequencing identified a heterozygous missense variant in patients’ RYR1 (19q13.2; NM_000540.2.14432C>T); however, its pathogenicity has remained uncertain. To determine the pathogenicity of this variant, we established a zebrafish (Danio rerio) model. Compared with avian and mammalian models, zebrafish provide several advantages for developmental genetic studies, including external fertilization and optical transparency during embryogenesis. Previous studies have demonstrated that zebrafish possess two ryr1 paralogs, ryr1a and ryr1b. Ryr1a is predominantly expressed in slow-twitch muscle fibers responsible for postural maintenance, whereas Ryr1b is mainly expressed in fast-twitch muscle fibers involved in rapid swimming movements. Loss of ryr1b function results in impaired SR Ca²⁺ release after 2 days post-fertilization, leading to defective skeletal muscle contraction and compromised locomotor performance. In this study, CRISPR interference (CRISPRi) was employed to knock down ryr1b by targeting its translation initiation site with guide RNAs, thereby mimicking RYR1 insufficiency. Combined with muscle function assessments and calcium imaging, this model was used to investigate the molecular mechanisms underlying the disease. Rescue assays were subsequently performed by introducing rabbit RYR1 mRNA carrying the patient-specific variant (RYR1-14429-SNV) to evaluate its functional effects. Muscle performance was assessed using escape response and swimming behavior assays. In the escape response assay, zebrafish larvae were stimulated on the snout using a plastic probe, and escape latency was recorded. The ryr1b knockdown group exhibited significantly prolonged escape times compared with the control group, which responded within an average of approximately 200 ms. Following rescue with RYR1-14429-SNV mRNA, escape performance improved in a dose-dependent manner but remained significantly impaired, indicating that the variant exerts a pathogenic effect on skeletal muscle contractility with a clear dose-dependent response. Correlation analysis between escape performance and quantitative PCR data further demonstrated a strong correlation (r = 0.9808), showing that reduced ryr1b expression was associated with prolonged escape latency. To further evaluate locomotor function, optovin-induced swimming assays were performed under ultraviolet light stimulation. Knockdown of ryr1b markedly reduced swimming angle, swimming frequency, and swimming endurance. Control larvae typically displayed tail-beat amplitudes of approximately ±20°, whereas the knockdown group showed a significant reduction to approximately ±8°. Rescue with RYR1-14429-SNV mRNA failed to restore locomotor function, as swimming frequency, tail-beat amplitude, and total swimming duration remained significantly lower than those of both the control and wild-type RYR1 rescue groups, closely resembling the knockdown phenotype. Furthermore, delayed initiation of swimming behavior suggested impaired Ca²⁺ mobilization. Calcium imaging further revealed that the RYR1-14429-SNV rescue group exhibited significant accumulation of Ca²⁺ within the SR, as indicated by increased red fluorescence intensity, whereas cytoplasmic free Ca²⁺ signals (green fluorescence) were significantly reduced compared with those of the control group. Quantitative analysis of the red-to-green fluorescence ratio demonstrated a dose-dependent reduction in Ca²⁺ mobilization from the SR to the cytoplasm. These findings suggest that the RYR1-14429 C>T variant impairs the Ca²⁺ channel function of RYR1, thereby reducing cytoplasmic Ca²⁺ availability required for actin–myosin interaction and skeletal muscle contraction. This defect provides a mechanistic explanation for the muscle weakness observed in both the escape response and swimming behavior assays, as well as for the delayed optovin-induced locomotor response. Collectively, our findings demonstrate that the RYR1-14429 C>T variant is pathogenic and most likely represents a loss-of-function mutation that compromises RYR1-mediated Ca²⁺ release, resulting in Ca²⁺ retention within the SR and insufficient cytoplasmic Ca²⁺ for effective muscle contraction. This mechanism may also explain the proximal muscle weakness observed in the affected family members. Finally, the ryr1b knockdown–rescue zebrafish model established in this study provides a robust platform for evaluating the pathogenicity of patient-specific RYR1 variants. This model may facilitate discrimination between gain-of-function mutations causing incomplete channel closure and loss-of-function mutations impairing Ca²⁺ conductance, thereby serving as a valuable platform for investigating the molecular mechanisms of RYR1-related myopathies. Moreover, this strategy supports the advancement of precision medicine while adhering to the 3Rs principles of animal research and may contribute to the future development and preclinical evaluation of targeted therapeutic strategies.