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首页> 外文期刊>Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration >Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay
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Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay

机译：使用双模式，来自Ninds收集的2095年ALS样品中C9ORF72己核苷酸重复区域的综合基因分型，使用两种模式，长读PCR测定

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Objective: Expansion of the G(4)C(2) repeat tract in the C9orf72 gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the C9orf72 repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (n = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. Methods: A single-tube 3-primer PCR assay mode, resolved using capillary electrophoresis, was used for sizing up to 145 repeats with single-repeat accuracy, for detecting expansions irrespective of their overall size, and for flagging confounding 3 ' sequence variations (SVs). A modified two-primer PCR mode, resolved via agarose gel electrophoresis, provided further size information for hyper-expanded samples (>145 repeats) up to similar to 5.8 kb amplicons (similar to 950 G(4)C(2) repeats). Results: Within the evaluated cohort, 177 (8.4%) samples were expanded, with 175 (99%) samples being hyper-expanded. 3 '-SVs were identified in 64 (3.1%) samples, and were most common in expanded alleles. Genotypes of all 606 (29%) homozygous samples were confirmed using an orthogonal PCR assay. Conclusion: This study and PCR method may improve and standardize molecular characterization of the C9orf72 locus, and have the potential to inform phenotype-genotype correlations and therapeutic development in ALS/FTD.
机译：目的：C9ORF72基因中的G（4）C（2）重复的膨胀与胎儿痴呆（FTD）和肌营养的外侧硬化（ALS）相关联。在这里，我们使用能够扩增富含GC的富含GC的序列的新型双峰PCR测定，提供全面的综合基因分型（N = 2095）（n = 2095）。方法：使用毛细管电泳分辨的单管3-底漆PCR测定模式用于尺寸高达145重复，以单重复精度，无论其整体尺寸如何，都可检测膨胀，以及用于标记混淆3'序列变化（ SVS）。通过琼脂糖凝胶电泳分辨的改性的双引物PCR模式，提供了与5.8kB扩增子相似的超膨胀样品（> 145重复）的进一步尺寸信息（类似于950g（4）C（2）重复）。结果：在评估的队列中，膨胀177（8.4％）样品，具有175（99％）样品的超膨胀。在64（3.1％）样品中鉴定了3'-SV，并且在扩展等位基因中最常见。使用正交PCR测定确认所有606（29％）纯合子样品的基因型。结论：该研究和PCR方法可以改善和标准化C9ORF72基因座的分子表征，并有可能在ALS / FTD中提供表型基因型相关性和治疗发育。

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