Chutake Yogesh K, Lam Christina, Costello Whitney N, Anderson Michael, Bidichandani Sanjay I
Department of Pediatrics, University of Oklahoma College of Medicine, Oklahoma City, OK.
Ann Neurol. 2014 Oct;76(4):522-8. doi: 10.1002/ana.24249. Epub 2014 Aug 30.
Friedreich ataxia (FRDA) is caused by an expanded GAA triplet-repeat (GAA-TR) mutation in the FXN gene. Patients are typically homozygous for expanded alleles containing 100 to 1,300 triplets, and phenotypic severity is significantly correlated with the length of the shorter of the 2 expanded alleles. Patients have a severe deficiency of FXN transcript, which is predominantly caused by epigenetic silencing of the FXN promoter. We sought to determine whether the severity of FXN promoter silencing is related to the length of the expanded GAA-TR mutation in FRDA.
Patient-derived lymphoblastoid cell lines bearing a range of expanded alleles (200-1,122 triplets) were evaluated for FXN transcript levels by quantitative reverse transcriptase polymerase chain reaction. FXN promoter function was directly measured by quantitative analysis of transcriptional initiation via metabolic labeling of newly synthesized transcripts in living cells.
FXN transcriptional deficiency was significantly correlated with the length of the shorter of the 2 expanded alleles, which was noted both upstream (R(2) = 0.84, p = 0.014) and downstream (R(2) = 0.89, p = 0.002) of the expanded GAA-TR mutation, suggesting that FXN promoter silencing in FRDA is related to repeat length. A bilinear regression model revealed that length dependence was strongest when the shorter of the 2 expanded alleles contained <400 triplets. Direct measurement of FXN promoter activity in patients with expanded alleles containing <400 versus >400 triplets in the shorter of the 2 expanded alleles revealed a significantly greater deficiency in individuals with longer GAA-TR alleles (p < 0.05).
FXN promoter silencing in FRDA is dependent on the length of the expanded GAA-TR mutation.
弗里德赖希共济失调（FRDA）由FXN基因中GAA三联体重复序列（GAA-TR）扩增突变引起。患者通常为含有100至1300个三联体的扩增等位基因的纯合子，且表型严重程度与两个扩增等位基因中较短者的长度显著相关。患者存在严重的FXN转录本缺陷，这主要是由FXN启动子的表观遗传沉默所致。我们试图确定FRDA中FXN启动子沉默的严重程度是否与扩增的GAA-TR突变的长度相关。
通过定量逆转录聚合酶链反应评估携带一系列扩增等位基因（200 - 1122个三联体）的患者来源的淋巴母细胞系中的FXN转录本水平。通过对活细胞中新合成转录本进行代谢标记来定量分析转录起始，从而直接测量FXN启动子功能。
FXN转录缺陷与两个扩增等位基因中较短者的长度显著相关，这在扩增的GAA-TR突变的上游（R² = 0.84，p = 0.014）和下游（R² = 0.89，p = 0.002）均有体现，表明FRDA中FXN启动子沉默与重复长度相关。双线性回归模型显示，当两个扩增等位基因中较短者包含<400个三联体时，长度依赖性最强。对两个扩增等位基因中较短者包含<400个与>400个三联体的患者的FXN启动子活性进行直接测量，结果显示GAA-TR等位基因较长的个体中缺陷明显更大（p < 0.05）。
FRDA中FXN启动子沉默取决于扩增的GAA-TR突变的长度。
