B4 Detection of the aberrantly spliced exon 1 - intron 1 htt mRNA in HD patient post mortem brain tissue and fibroblast lines
B4 Detection of the aberrantly spliced exon 1 - intron 1 htt mRNA in HD patient post mortem brain tissue and fibroblast lines
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B4 HD 患者死后脑组织和成纤维细胞系中异常剪接的外显子 1 - 内含子 1 htt mRNA 的检测
DOI：
10.1136/jnnp-2016-314597.35
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Bates G
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作者：
Bates G
背景我们先前已经证明亨廷顿基因的外显子1并不总是剪接到外显子2，导致产生编码高致病性外显子1 HTT蛋白的小聚腺苷酸化mRNA。该通读产物的水平与CAG重复长度成比例，并存在于所有HD敲入小鼠模型（CAG ≥ 50）中，利用位于小鼠内含子1中677 bp和1145 bp处的隐蔽聚腺苷酸化位点。在YAC 128和BACHD小鼠模型中也容易检测到通读产物，这两种小鼠模型都使用位于人内含子1中7327 bp的隐蔽聚腺苷酸化位点。然而，这种小的外显子1 -内含子1 mRNA的存在是不容易检测到HD患者组织中使用的测定，我们最初developed.AimsTo开发新的定量RT-PCR检测方法来检测外显子1 -内含子1 HTT mRNA在HD patients.MethodsWe组织中现在已经建立了一套qPCR检测，定量序列位于接近隐藏的多聚腺苷酸化位点在人类HTT内含子1。这些已被应用到一系列的成纤维细胞系和死后的大脑样本从个人与成人发病或青少年发病HD.ResultsThe外显子1 -内含子1 HTT mRNA可以很容易地检测到在躯体感觉皮层，海马和小脑的死后的大脑从个人与HD，特别是在那些早发性疾病。这些人HTT内含子序列也存在于来自青少年HD患者的成纤维细胞中。我们还将提供关于基因敲入小鼠模型中外显子1 -内含子1 HTT mRNA相对丰度随疾病进展而变化的数据。ConclusionThe highly pathogenic exon 1 HTT protein is generated in the tissues of people with HD through the aberrant splicing of HTT.FundingMedical Research理事会and CHDI Foundation
BackgroundWe have previously shown that exon 1 of the huntingtin gene does not always splice to exon 2 resulting in the production of a small polyadenylated mRNA that encodes the highly pathogenic exon 1 HTT protein. The level of this read-through product is proportional to CAG repeat length and present in all knock-in mouse models of HD (with CAG ≥ 50), utilising cryptic polyadenylation sites located at 677 bp and 1145 bp into mouse intron 1. The read-through product is also readily detected in the YAC128 and BACHD mouse models which both use a cryptic polyadenylation site that is located 7327 bp into human intron 1. However, the presence of this small exon 1 – intron 1 mRNA was not easy to detect in HD patient tissues using the assays that we initially developed.AimsTo develop novel quantitative RT-PCR assays to detect the exon 1 – intron 1 HTT mRNA in tissues from HD patients.MethodsWe have now established a set of qPCR assays that quantify sequences located close to the cryptic polyadenylation site in human HTT intron 1. These have been applied to a series of fibroblast lines and post-mortem brain samples from individuals with either adult-onset or juvenile-onset HD.ResultsThe exon 1 – intron 1 HTT mRNA can be readily detected in the somatosensory cortex, hippocampus and cerebellum of post mortem brains from individuals with HD, particularly in those with early onset disease. These human HTT intronic sequences are also present in fibroblasts from juvenile HD patients. We shall also present data on changes in the relative abundance of the exon 1 – intron 1 Htt mRNA in a knock-in mouse model with disease progression.ConclusionThe highly pathogenic exon 1 HTT protein is generated in the tissues of people with HD through the aberrant splicing of HTT.FundingMedical Research Council and CHDI Foundation