PDF(Pubmed)
Abstract：
Cerebellar ataxia, neuropathy and vestibular areflexia syndrome is a progressive, generally late-onset, neurological disorder associated with biallelic pentanucleotide expansions in Intron 2 of the RFC1 gene. The locus exhibits substantial genetic variability, with multiple pathogenic and benign pentanucleotide repeat alleles previously identified. To determine the contribution of pathogenic RFC1 expansions to neurological disease within an Australasian cohort and further investigate the heterogeneity exhibited at the locus, a combination of flanking and repeat-primed PCR was used to screen a cohort of 242 Australasian patients with neurological disease. Patients whose data indicated large gaps within expanded alleles following repeat-primed PCR, underwent targeted long-read sequencing to identify novel repeat motifs at the locus. To increase diagnostic yield, additional probes at the RFC1 repeat region were incorporated into the PathWest diagnostic laboratory targeted neurological disease gene panel to enable first-pass screening of the locus for all samples tested on the panel. Within the Australasian cohort, we detected known pathogenic biallelic expansions in 15.3% (n = 37) of patients. Thirty indicated biallelic AAGGG expansions, two had biallelic \'Māori alleles\' [(AAAGG)exp(AAGGG)exp], two samples were compound heterozygous for the Māori allele and an AAGGG expansion, two samples had biallelic ACAGG expansions and one sample was compound heterozygous for the ACAGG and AAGGG expansions. Forty-five samples tested indicated the presence of biallelic expansions not known to be pathogenic. A large proportion (84%) showed complex interrupted patterns following repeat-primed PCR, suggesting that these expansions are likely to be comprised of more than one repeat motif, including previously unknown repeats. Using targeted long-read sequencing, we identified three novel repeat motifs in expanded alleles. Here, we also show that short-read sequencing can be used to reliably screen for the presence or absence of biallelic RFC1 expansions in all samples tested using the PathWest targeted neurological disease gene panel. Our results show that RFC1 pathogenic expansions make a substantial contribution to neurological disease in the Australasian population and further extend the heterogeneity of the locus. To accommodate the increased complexity, we outline a multi-step workflow utilizing both targeted short- and long-read sequencing to achieve a definitive genotype and provide accurate diagnoses for patients.
摘要：
小脑共济失调,神经病变和前庭反射综合征是一种进行性,通常是迟发性的，与RFC1基因内含子2双等位基因五核苷酸扩增相关的神经系统疾病。该基因座表现出大量的遗传变异，具有先前鉴定的多个致病性和良性五核苷酸重复等位基因。为了确定澳大利亚队列中致病性RFC1扩张对神经系统疾病的贡献，并进一步调查该位点表现出的异质性，我们使用侧翼PCR和重复引物PCR的组合筛选了242例澳大利亚神经系统疾病患者的队列.重复引物PCR后数据显示扩展等位基因内存在较大差距的患者，进行了靶向长读测序，以鉴定基因座处的新重复基序。为了提高诊断产量，我们在PathWest诊断实验室靶向神经疾病基因小组中纳入了RFC1重复区的额外探针,以便对小组中所有检测样本进行首过位点筛查.在澳大利亚队列中，我们在15.3%(n=37)的患者中检测到已知的致病性双等位基因扩增。三十种指示的双等位基因AAGGG扩增，两个人具有双等位基因\'毛利人等位基因\'[（AAAGG）exp（AAGGG）exp]，两个样本是毛利人等位基因和AAGGG扩增的复合杂合，两个样品具有双等位基因ACAGG扩增,一个样品是ACAGG和AAGGG扩增的复合杂合。测试的45个样品表明存在未知致病性的双等位基因扩增。大部分（84％）在重复引发的PCR后显示出复杂的中断模式，这表明这些扩展可能由不止一个重复图案组成，包括以前未知的重复。使用靶向长读测序，我们在扩展的等位基因中鉴定了三个新的重复基序。这里,我们还表明,短读序列测序可用于可靠地筛选使用PathWest靶向神经系统疾病基因组测试的所有样本中是否存在双等位基因RFC1扩增.我们的结果表明，RFC1致病性扩展对澳大利亚人群的神经系统疾病做出了重大贡献，并进一步扩大了基因座的异质性。为了适应增加的复杂性，我们概述了一个多步骤的工作流程,利用有针对性的短阅读和长阅读测序来获得明确的基因型,并为患者提供准确的诊断.
