Tissue-specific reduction in splicing efficiency of IKBKAP due to the major mutation associated with familial dysautonomia
Tissue-specific reduction in splicing efficiency of IKBKAP due to the major mutation associated with familial dysautonomia
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DOI：
10.1086/368263
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发表时间：
2003-03-01
影响因子：
9.8
通讯作者：
Slaugenhaupt, SA
中科院分区：
文献类型：
--
作者：
Cuajungco, MP;Leyne, M;Slaugenhaupt, SA
我们最近确定了I-kappaB激酶相关蛋白（IKBKAP）基因突变是家族性自主神经功能障碍（FD）的主要原因，FD是一种隐性感觉和自主神经病变。这种改变位于内含子20供体剪接位点的碱基对6，存在于>99.5%的FD染色体上，并导致外显子20的组织特异性跳跃。第二个FD突变，外显子19（R696 P）的错义变化，仅见于4例杂合子的主要突变。在此，我们通过检测EBV转化的淋巴母细胞系、原代成纤维细胞、新鲜采集的血液样本和FD患者的尸检组织中野生型与突变型（WT：MU）IKBKAP转录本的比例，进一步表征了主要突变的后果。我们一致地发现，WT IKBKAP转录本存在于所有细胞系、血液和死后FD组织中，尽管程度不同。此外，在FD细胞系和组织中观察到WT蛋白水平的相应降低。培养的淋巴母细胞中WT：MU的比例随生长阶段而变化，但不随血清浓度或抗生素的加入而变化。使用光密度和实时定量聚合酶链反应，我们发现，相对WT：MU IKBKAP RNA水平最高的培养患者的淋巴母细胞和最低的死后中枢和周围神经组织。这些观察结果表明，WT IKBKAP mRNA生产的相对低效的突变等位基因在神经系统中的FD感觉和自主神经元的选择性变性的基础。因此，探索增加神经系统中WT：MU IKBKAP转录物比例的方法为开发FD患者的有效治疗提供了有希望的方法。
We recently identified a mutation in the I-kappaB kinase associated protein (IKBKAP) gene as the major cause of familial dysautonomia (FD), a recessive sensory and autonomic neuropathy. This alteration, located at base pair 6 of the intron 20 donor splice site, is present on >99.5% of FD chromosomes and results in tissue-specific skipping of exon 20. A second FD mutation, a missense change in exon 19 (R696P), was seen in only four patients heterozygous for the major mutation. Here, we have further characterized the consequences of the major mutation by examining the ratio of wild-type to mutant (WT: MU) IKBKAP transcript in EBV-transformed lymphoblast lines, primary fibroblasts, freshly collected blood samples, and postmortem tissues from patients with FD. We consistently found that WT IKBKAP transcripts were present, albeit to varying extents, in all cell lines, blood, and postmortem FD tissues. Further, a corresponding decrease in the level of WT protein is seen in FD cell lines and tissues. The WT: MU ratio in cultured lymphoblasts varied with growth phase but not with serum concentration or inclusion of antibiotics. Using both densitometry and real-time quantitative polymerase chain reaction, we found that relative WT: MU IKBKAP RNA levels were highest in cultured patient lymphoblasts and lowest in postmortem central and peripheral nervous tissues. These observations suggest that the relative inefficiency of WT IKBKAP mRNA production from the mutant alleles in the nervous system underlies the selective degeneration of sensory and autonomic neurons in FD. Therefore, exploration of methods to increase the WT: MU IKBKAP transcript ratio in the nervous system offers a promising approach for developing an effective therapy for patients with FD.