An intronic bi-allelic pentanucleotide repeat expansion mutation, (AAGGG)_400–2000, at AAAAG repeat locus in RFC1 gene, is known as underlying genetic cause in cases with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) and late-onset sporadic ataxia. Biallelic positive cases carry a common recessive risk haplotype, “AAGA,” spanning RFC1 gene. In this study, our aim is to find prevalence of bi-allelic (AAGGG)_exp in Indian ataxia and other neurological disorders and investigate the complexity of RFC1 repeat locus and its potential association with neurodegenerative diseases in Indian population-based cohorts. We carried out repeat number and repeat type estimation using flanking PCR and repeat primed PCR (AAAAG/AAAGG/AAGGG) in four Indian disease cohorts and healthy controls. Haplotype assessment of suspected cases was done by genotyping and confirmed by Sanger sequencing. Blood samples and consent of all the cases and detailed clinical details of positive cases were collected in collaboration with A.I.I.M.S. Furthermore, comprehension of RFC1 repeat locus and risk haplotype analysis in Indian background was performed on the NGS data of Indian healthy controls by ExpansionHunter, ExpansionHunter Denovo, and PHASE analysis, respectively. Genetic screening of RFC1-TNR locus in 1998 uncharacterized cases (SCA12: 87; uncharacterized ataxia: 1818, CMT: 93) and 564 heterogenous controls showed that the frequency of subjects with bi-allelic (AAGGG)_exp are 1.15%, < 0.05%, 2.15%, and 0% respectively. Two RFC1 positive sporadic late-onset ataxia cases, one bi-allelic (AAGGG)_exp and another, (AAAGG)_~700/(AAGGG)_exp, had recessive risk haplotype and CANVAS symptoms. Long normal alleles, 15–27, are significantly rare in ataxia cohort. In IndiGen control population (IndiGen; N = 1029), long normal repeat range, 15–27, is significantly associated with A₃G₃ and some rare repeat motifs, AGAGG, AACGG, AAGAG, and AAGGC. Risk-associated “AAGA” haplotype of the original pathogenic expansion of A₂G₃ was found associated with the A₃G₃ representing alleles in background population. Apart from bi-allelic (AAGGG)_exp, we report cases with a new pathogenic expansion of (AAAGG)_exp/(AAGGG)_exp in RFC1 and recessive risk haplotype. We found different repeat motifs at RFC1 TNR locus, like AAAAG, AAAGG, AAAGGG, AAAAGG, AAGAG, AACGG, AAGGC, AGAGG, and AAGGG, in Indian background population except ACAGG and (AAAGG)_n/(AAGGG)_n. Our findings will help in further understanding the role of long normal repeat size and different repeat motifs, specifically AAAGG, AAAGGG, and other rare repeat motifs, at the RFC1 locus.

Graphical Abstract

中文翻译：

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印度队列中不同神经退行性结果中 RFC1 串联核苷酸重复位点的研究

RFC1基因AAAAG 重复位点上的内含子双等位基因五核苷酸重复扩展突变 (AAGGG) _400-2000被认为是小脑性共济失调、神经病、前庭无反射综合征 (CANVAS) 和晚发型病例的潜在遗传原因。散发性共济失调。双等位基因阳性病例携带常见的隐性风险单倍型“AAGA”，涵盖RFC1基因。在这项研究中，我们的目的是发现印度共济失调和其他神经系统疾病中双等位基因 (AAGGG) _{exp的患病率，并研究}RFC1重复基因座的复杂性及其与印度人群中神经退行性疾病的潜在关联。我们使用侧翼 PCR 和重复引物 PCR (AAAAG/AAAGG/AAGGG) 在四个印度疾病队列和健康对照中进行重复次数和重复类型估计。通过基因分型对疑似病例进行单倍型评估，并通过桑格测序进行确认。与 AIIMS 合作收集所有病例的血样和同意书以及阳性病例的详细临床细节 此外，通过ExpansionHunter、ExpansionHunter Denovo 对印度健康对照的 NGS 数据进行了印度背景RFC1重复基因座的理解和风险单倍型分析，和相位分析，分别。对 1998 个无特征病例（SCA12：87；无特征共济失调：1818，CMT：93）和 564 个异质对照进行RFC1- TNR 位点基因筛查，结果显示双等位基因（AAGGG）exp 受试者的频率为 1.15%，< _0.05 % 、 2.15% 和 0% 分别。两例RFC1阳性散发性迟发性共济失调病例，一例为双等位基因 (AAGGG) _exp，另一例为 (AAAGG) _~700 /(AAGGG) _exp，具有隐性风险单倍型和 CANVAS 症状。长的正常等位基因（15-27）在共济失调队列中非常罕见。在 IndiGen 对照群体（IndiGen；N = 1029）中，长正常重复范围（15-27）与 A ₃ G ₃和一些罕见的重复基序 AGAGG、AACGG、AAGAG 和 AAGGC 显着相关。发现A ₂ G ₃原始致病性扩展的风险相关“AAGA”单倍型与代表背景人群中的等位基因的A ₃ G _3相关。除了双等位基因 (AAGGG) _{exp之外，我们还报告了}RFC1中具有新致病性扩展 (AAAGG) _exp /(AAGGG) _exp和隐性风险单倍型的病例。我们在RFC1中发现了不同的重复基序印度背景人群中的 TNR 位点，如 AAAAG、AAAGG、AAAGGG、AAAAGG、AAGAG、AACGG、AAGGC、AGAGG 和 AAGGG，除了 ACAGG 和 (AAAGG) _n /(AAGGG) _n之外。我们的研究结果将有助于进一步了解RFC1基因座上长正常重复大小和不同重复基序（特别是 AAAGG、AAAGGG 和其他罕见重复基序）的作用。

图形概要