本論文的目的在探討台灣三個 Romano-Ward Long-QT 徵候群 (LQTS) 家族的分子致因。LQTS 是心臟猝死症的一種，為體染色體顯性的遺傳性疾病，患者因心肌再極化的異常，臨床上出現心電圖之 QT interval 延長、T wave 不正常以及常患有心律不整、暈厥、突然死亡等症狀。LQTS 在遺傳上是異質性的，已有四個和此疾病相關的心肌離子通道基因的突變被報導，包括染色體 11p15.5 上的 LQT1 (KVLQT1，IKs 的 K+ 通道基因)、染色體 7q35-36 上的 LQT2 (HERG，IKr 的 K+ 通道基因)、染色體 3p21-24 上的 LQT3 (SCN5A，Na+ 通道基因)、及染色體 21q22.1-22.2 上的 LQT5 (KCNE1，K+ 離子通道的調節基因)。第五個基因是染色體 4q25-27 上的 LQT4 ，目前尚未被選殖定序出來；且至少還有一種未被定位的基因與本疾病相關。
本論文所探討的 LQTS 家族 H87、C86 由林口長庚紀念醫院及台北榮民總醫院提供，L85 家族由苗栗為恭紀念醫院提供，並皆完成外表性狀分析。家族 C86 先前已經連鎖分析排除為 LQT1，家族 L85 綜合先前經連鎖分析的結果及臨床上的表現，推測為和 HERG 連鎖的 LQT2。本論文首先對 H87 及 C86 家族進行鄰近各 LQTS 基因座的多型性標記之連鎖分析，確定 H87 家族患者為 LQT2，C86 家族不連鎖於 LQT1～5。接著利用聚合酵素鏈反應 (PCR)、單股核酸構形多型性 (SSCP) 及 DNA 定序來檢視 H87 及 L85 家族的 HERG 基因突變。H87 家族的突變分析結果發現 HERG cDNA 第 2230 個核甘酸發生 C*T 的轉換 (transition)，使 HERG 第 744 個精胺酸 (arginine) 改變為一終止密碼 (即 R744X)，此突變所合成的蛋白質缺少了環狀核甘酸 (cAMP) 結合部位及碳 (C) 端的胺基酸。此核甘酸變異產生了一新的限制 DdeI 的切割位置，故以此限制的切割進一步檢視 H87 家族的 HERG 基因，結果發現此家族患者皆比正常個體多了一切割位置。家族 L85 經突變分析結果發現 HERG cDNA 第 1907 個核甘酸發生 C*T 的轉換，使 HERG 第 614 個胺基酸由丙胺酸 (alanine) 轉變為頡胺酸 (valine) (即 A614V)，此胺基酸位於離子通道的孔道部位 (pore domain)。此核甘酸變異產生了一新的限制 Bsp1286I 的切點，故以此限制的切割進一步檢視 L85 家族的 HERG 基因，結果發現患者的父親及弟弟皆有 A614V 突變。由於患者的父親與叔父均遺傳了祖父的同一對偶基因，然而叔父並不具 A614V 突變，故推測此為一新突變 (de novo mutation)。家族致病突變基因的發現及發展出的突變基因檢測試驗，可為患者家族提供症狀出現前 (pre-symptomatic) 或出生前 (prenatal) 的檢測，以有利於患者家族。

Romano-Ward Long-QT syndrome (LQTS) is an autosomal dominant disorder of cardiac repolarization that results in prolonged QT interval and T wave abnormalities on the electrocardiographs, ventricular arrhythmia, syncope and sudden death. The inherited basis of LQTS is heterogeneous. Five loci and four genes responsible for LQTS have been identified. The LQT1 locus (chromosome 11p15.5) encodes the KVLQT1 K+ channel, which co-assembles with IsK protein (encoded by LQT5 locus, chromosome 21q22.1-22.2) to form the slowly activating delayed rectifier IKs current. The LQT2 locus (chromosome 7q35-36) encodes the HERG K+ channel, which underlies the rapidly activating current IKr. The LQT3 locus (chromosome 3p21-24) encodes the cardiac Na+ channel, SCN5A. A number of heterozygous mutations have been described for each gene. The LQT4 locus is on chromosome 4q25-27, but the gene has not been isolated. Since there are other families with the long QT phenotype not linking to any of these gene loci, one or more genes may exist.
The purpose of this study was to examine the molecular basis of LQTS in three Taiwanese families. Families H87 and C86 were provided by Chang Gung Memorial Hospital and Veterans General Hospital-Taipei. Family C86 was excluded from linkage at LQT1 previously. Family L85 was provided by Wei-Gong Memorial Hospital and was established linkage at LQT2 previously. Families H87 and C86 were genotyped with polymorphic makers corresponding to the LQTS loci. No evidence for linkage of disease phenotype was observed in family C86. Conversely, the disease cosegregated with three LQT2 makers in family H87. To test the hypothesis that HERG is the gene responsible for LQTS in families H87 and L85, the coding sequences of HERG were amplified and screened by single strand conformation polymorphism (SSCP) for mutation. A C to T transition at the first nucleotide of codon 744, resulting in the substitution of arginine for termination codon (R744X) was identified in family H87. The novel R744X mutation is predicted to result in truncation of the putative cyclic nucleotide-binding domain (NBD) and C-terminal region of the protein. The mutation was confirmed by DdeI endonuclease digestion and was present in all affected family members. In family L85, a recurrent A614V mutation in the pore region of HERG was identified. The mutation was the result of C to T transition changed the codon 614 from alanine to valine. By Bsp1286I endonuclease digestion, the mutation was present in all affected family members. Since the unaffected paternal uncle inherited the same allele form the grandfather as the proband*s father, a de novo mutation apparently occurred in the father and transmitted to his offspring. Identification of mutation can not only offer pre-symptomatic or prenatal genetic diagnosis, but also elucidate the possibility of new therapeutic approaches for treatment and prevention of this cardiovascular disease.

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