遗传性 Transthyretin Ala97Ser 相关淀粉样变性的遗传分析
1Department of Traditional Chinese Medicine, Division of Chinese Acupuncture and Traumatology,Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine, 2Department of Neurology,Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine
Summary
在这里, 我们提出了一个协议, 以确认存在的点突变诊断遗传性 transthyretin 淀粉样变性, 使用 Ala97Ser, 最常见的地方性突变在台湾, 作为一个例子。
Abstract
遗传检测是遗传性 transthyretin 相关淀粉样变性的最可靠的试验, 应在 transthyretin 淀粉样变性 (ATTR) 的大多数病例中进行。ATTR 是一种罕见的, 但致命的疾病与异构表型;因此, 诊断有时会延迟。因此, 随着对 ATTR 早期表现的关注和广泛的认识, 以及新的治疗方法, 适当的诊断研究, 包括 transthyretin (TTR) 基因检测, 以确认 ATTR 的类型和变种, 因此改善预后的基础。利用聚合酶链反应 (PCR) 方法进行遗传分析, 证实 TTR 点突变比传统方法 (如南方印迹) 的存在更为迅速和安全。在此, 我们展示了基因确认的 ATTR Ala97Ser 突变, 最常见的地方性突变在台湾。该协议包括四个主要步骤: 收集全血标本, dna 提取, 所有四 TTR 外显子的基因分析与 PCR, 和 DNA 测序。
Introduction
Transthyretin (TTR) 淀粉样变性 (ATTR) 是遗传性系统性淀粉样变性1的最常见形式, 可由 Transthyretin (TTR) 基因2中的常染色体显性遗传突变引起。TTR 突变破坏 tetrameric 蛋白结构的稳定, 并导致其离解成单体, 重组成淀粉样纤维2。超过 100 amyloidogenic TTR 突变已被报告全球1。用聚合酶链反应 (PCR) 方法进行遗传分析, 证实了 TTR 点突变的存在, 具有避免放射性标记探针处理与南方印迹3相比较的优点。PCR 是一种快速、简便、廉价、可靠的技术, 已应用于现代科学的众多领域4。
由于其表型异质性, 早期诊断这一进步和致命的疾病是有挑战性的。随着对 ATTR 早期表现的关注和广泛的认识, 以及新出现的治疗方法5, 适当的诊断研究, 包括 TTR 基因检测, 对于改善预后至关重要。此外, 不同的突变与不同显性的特点, 发病年龄, 进展模式, 疾病严重性, 中位生存率, 肝移植的疗效, 或 TTR 稳定剂2,6, 和不同程度的神经和心脏参与, 这对基因咨询有很大的影响7,8,9。此外, 高度准确的基因检测是唯一的工具, 区分两种不同类型的 ATTR: 遗传性 (突变) 和野生类型 (非突变体, 老年系统性淀粉样变性, SSA)7。必须确认 ATTR 的类型, 因为治疗方法相差很大2。因此, TTR 遗传试验的逐步性协议的描述越来越有必要。
以台湾最常见的地方性突变 Ala97Ser 为例, 说明了检测突变的分子方法。dna 提取步骤的修改减少了三种溶液的用量, 并产生了足够数量的 dna。在该协议中, 对所有四 TTR 外显子进行了分析, 而包括 5 ‘ 上游、3 ‘ 下游、启动子、内含子和未翻译区域 (UTR) 的区域未进行排序。
Protocol
在实验室进行的测试是根据1988年《临床实验室改善修订》 (CLIA) 的要求而进行的, 由昌热心纪念医院机构评审委员会批准的规例及大学 (执照100-4470A3 和 104-2462A3)。所有患者均获得知情同意。 1. 采血标本收集 将全血收集到商用的 EDTA 处理管中。轻轻混合, 将血液样本储存在4摄氏度, 直到加工。 2. 从外周血中提取 DNA 使用 dna ?…
Representative Results
琼脂糖凝胶电泳两个病人和一个健康的个人揭示了预期大小的带, 包括 454 bp PCR 产品的外显子4的 TTR 基因 (图 1)。 图 1.凝胶电泳描述 PCR 扩增 TTR 基因.正常: 健康的个体。车道 M: 100 bp DNA 梯子作为分子大小标?…
Discussion
协议中有两个关键步骤。首先, 为了有足够数量的白细胞, 应避免 hemodiluted 标本11。其次, 使用合适的 PCR 引物是取得可靠结果12的根本。我们使用的底漆爆破 web 工具设计引物4,13;在四 TTR 外显子的每一侧至少有40基对。我们还在 NCBI 上进行爆炸检查底漆的特异性。
在 DNA 提取步骤中进行了一些修改?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢新趣吴小姐在实验中的帮助。这项研究得到了长功夫医学研究计划 (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) 和 100-4470A3, 104-2462A3, 台湾的资助。
Materials
| EDTA-treated tubes | BD | ||
| DNA Extraction Kit | Stratagen | 200600 | |
| NanoDrop ND2000 spectrophotometer | Thermo Fisher Scientific | NanoDrop 2000 | |
| Delicate Task Wipers | Kimberly-Clark | Kimtech Science Kimwipes | |
| AmpliTaq Gold 360 DNA Polymerase kit | Applied Biosystems | 4398823 | |
| TTR gene intronic primers | Exon1 | F: 5’-TCAGATTGGCAGGGATAAGC-3’ | |
| Exon1 | R: 5’-GCAAAGCTGGAAGGAGTCAC-3’ | ||
| Exon2 | F: 5’-TCTTGTTTCGCTCCAGATTTC-3’ | ||
| Exon2 | R: 5’-TCTACCAAGTGAGGGGCAAA-3’ | ||
| Exon3 | F: 5’-GTGTTAGTTGGTGGGGGTGT-3’ | ||
| Exon3 | R: 5’-TGAGTAAAACTGTGCATTTCCTG-3’ | ||
| Exon4 | F: 5’-GACTTCCGGTGGTCAGTCAT-3’ | ||
| Exon4 | R: 5’-GCGTTCTGCCCAGATACTTT-3’ | ||
| thermocycler | Applied Biosystems | GeneAmp PCR System 9700 | |
| electrophoresis cell | ADVANCE | Mupid-2plus | |
| DNA ladder | Protech | PT-M1-100 | |
| dye | BioLabs | B7021 | |
| AlphaImager EC | Alpha Innotech | AlphaImager EC | |
| automatic sequencer | Applied Biosystems | 3730xl DNA Analyzer |
Referências
- Planté-Bordeneuve, V., Said, G. Familial amyloid polyneuropathy. The Lancet Neurology. 10 (12), 1086-1097 (2011).
- Planté-Bordeneuve, V., et al. Long-term treatment of transthyretin familial amyloid polyneuropathy with tafamidis: a clinical and neurophysiological study. Journal of Neurology. 264 (2), 268-276 (2017).
- Nichols, W. C., Benson, M. D. Hereditary amyloidosis: detection of variant prealbumin genes by restriction enzyme analysis of amplified genomic DNA sequences. Clinical Genetics. 37 (1), 44-53 (1990).
- Lorenz, T. C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. Journal of Visualized Experiments. (63), e3998 (2012).
- Hsu, H. C., et al. Phenotypic expressions of hereditary Transthyretin Ala97Ser related Amyloidosis (ATTR) in Taiwanese. BMC Neurology. 17 (1), 178 (2017).
- Barroso, F. A., et al. Long-term safety and efficacy of tafamidis for the treatment of hereditary transthyretin amyloid polyneuropathy: results up to 6 years. Amyloid. 24 (3), 194-204 (2017).
- Rapezzi, C., et al. Disease profile and differential diagnosis of hereditary transthyretin-related amyloidosis with exclusively cardiac phenotype: an Italian perspective. European Heart Journal. 34 (7), 520-528 (2013).
- Mariani, L. L., et al. Genotype-phenotype correlation and course of transthyretin familial amyloid polyneuropathies in France. Annals of Neurology. 78 (6), 901-916 (2015).
- Hammarström, P., Jiang, X., Hurshman, A. R., Powers, E. T., Kelly, J. W. Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity. Proceedings of the National Academy of Sciences of the United States of America. 99, 16427-16432 (2002).
- Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S., Madden, T. L. NCBI BLAST: a better web interface. Nucleic Acids Research. 36, 5-9 (2008).
- Fox, A. J., et al. Next generation sequencing for the detection of actionable mutations in solid and liquid tumors. Journal of Visualized Experiments. (115), (2016).
- Date, Y., Nakazato, M., Kangawa, K., Shirieda, K., Fujimoto, T., Matsukura, S. Detection of three transthyretin gene mutations in familial amyloidotic polyneuropathy by analysis of DNA extracted from formalin-fixed and paraffin-embedded tissues. Journal of the Neurological Sciences. 150 (2), 143-148 (1997).
- Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T. L. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13, 134 (2012).
- Green, M. R., Sambrook, J. . Molecular cloning: A laboratory manual. , (2012).
- Buckingham, L., Flaws, M. L. . Molecular diagnostics: Fundamentals, methods, and clinical applications. , (2012).
- Tan, S. C., Yiap, B. C. DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology. 2009, (2009).
- Rapezzi, C., et al. Systemic cardiac amyloidoses: disease profiles and clinical courses of the 3 main types. Circulation. 120 (13), 1203-1212 (2009).
- Gertz, M. A., Dispenzieri, A., Sher, T. Pathophysiology and treatment of cardiac amyloidosis. Nature Reviews Cardiology. 12 (2), 91-102 (2015).
- Vrana, J. A., et al. Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics. Haematologica. 99 (7), 1239-1247 (2014).
- Nakamura, M., et al. Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay. Human Heredity. 49 (4), 186-189 (1999).
- Ueda, M., Ando, Y. Recent advances in transthyretin amyloidosis therapy. Translational Neurodegeneration. 3, 19 (2014).
- Brown, E. E., et al. Genetic testing improves identification of transthyretin amyloid (ATTR) subtype in cardiac amyloidosis. Amyloid. 24 (2), 92-95 (2017).
- Tasaki, M., et al. Rapid detection of wild-type and mutated transthyretins. Annals of Clinical Biochemistry. 53 (4), 508-510 (2015).
- Plante-Bordeneuve, V., et al. Diagnostic pitfalls in sporadic transthyretin familial amyloid polyneuropathy (TTR-FAP). Neurology. 69 (7), 693-698 (2007).
- Hsu, J. L., et al. A prospective, observational study of patients with uncommon distal symmetric painful small-fiber neuropathy. PLoS ONE. 12 (9), 0183948 (2017).
- Salvi, F., Pastorelli, F., Plasmati, R., Bartolomei, I., Dall’Osso, D., Rapezzi, C. Genotypic and phenotypic correlation in an Italian population of hereditary amyloidosis TTR-related (HA-TTR): Clinical and neurophysiological aids to diagnosis and some reflections on misdiagnosis. Amyloid. 19, 58-60 (2012).
- Suhr, O. B., Lundgren, E., Westermark, P. One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition. Journal of Internal Medicine. 281 (4), 337-347 (2017).
- Vilà-Rico, M., et al. Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS. Journal of Proteomics. 127, 234-246 (2015).
Citar este artigo
Hsu, H., Liao, M., Hsu, J., Lee, Y., Ro, L. Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis. J. Vis. Exp. (136), e57743, doi:10.3791/57743 (2018).