识别RP1-Alu插入的诊断挑战及其克服策略-动脉网

Identification of diagnostic challenges in RP1 Alu insertion and strategies for overcoming them

Nature 等信源发布 2024-10-24 15:59

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AbstractRecently, a founder Alu insertion in exon 4 of RP1 was detected in Japanese and Korean patients with inherited retinal diseases (IRDs). However, carrier frequency and diagnostic challenges for detecting AluY insertion are not established. We aim to investigate the frequency of AluY in individuals with or without IRDs and to overcome common diagnostic pitfalls associated with AluY insertion.

摘要最近，在日本和韩国遗传性视网膜疾病（IRD）患者中检测到RP1外显子4的创始人Alu插入。但是，尚未确定检测AluY插入的载波频率和诊断挑战。我们的目标是调查有或没有IRD的个体中AluY的频率，并克服与AluY插入相关的常见诊断缺陷。

A total of 1,072 subjects comprising 411 patients with IRD (IRD group) and 661 patients with other suspected Mendelian genetic disease (non-IRD group) was screened for AluY insertion. Targeted panel sequencing and whole-genome sequencing were used for detection of AluY insertion, and an optimized allele-specific PCR (AS-PCR) was used for validation.

筛选了总共1072名受试者，其中包括411名IRD患者（IRD组）和661名其他疑似孟德尔遗传病患者（非IRD组）。靶向面板测序和全基因组测序用于检测AluY插入，并使用优化的等位基因特异性PCR（AS-PCR）进行验证。

The AluY insertion was detected in 1.5% in IRD group (6/411). The AluY insertion was not observed in non-IRD group (0/661). All patients with AluY were confirmed to have RP1 pathogenic variants on the paired allele. We identified AluY allele dropout leading to false homozygosity for c.4196del pathogenic variant in Sanger sequencing.

IRD组的AluY插入率为1.5%（6/411）。在非IRD组（0/661）中未观察到AluY插入。所有AluY患者均被证实在配对等位基因上具有RP1致病变异。我们在Sanger测序中鉴定出AluY等位基因缺失导致c.4196del致病变体的假纯合性。

The allelic relationship between variants of RP1 was accurately determined by AluY AS-PCR. Delineating diagnostic challenges of AluY insertion and strategies to avoid potential pitfalls could aid clinicians in an accurate molecular diagnosis for patients with IRD..

通过AluY AS-PCR准确确定了RP1变体之间的等位基因关系。描述AluY插入的诊断挑战和避免潜在陷阱的策略可以帮助临床医生对IRD患者进行准确的分子诊断。。

IntroductionInherited retinal diseases (IRDs) are a group of diverse genetic disorders associated with visual impairment due to progressive degeneration of the retina and affect more than 2 million people worldwide1,2. Genetic diagnosis of IRDs is particularly challenging and often delayed due to the extensive genetic heterogeneity and overlapping, variable, and incompletely penetrant nature of the clinical presentation1,2.

引言遗传性视网膜疾病（IRD）是一组与视网膜进行性变性引起的视力障碍相关的多种遗传疾病，影响全球200多万人1,2。IRD的遗传诊断特别具有挑战性，并且由于广泛的遗传异质性以及临床表现的重叠，可变和不完全渗透性质而经常被延迟1,2。

To date, more than 280 genes have been identified as responsible for IRD development, with either autosomal dominant or recessive or X-linked inheritance patterns (RetNet, https://sph.uth.edu/retnet/ last accessed on February 2024). Retinitis pigmentosa (RP, MIM #268000) is the most common IRD, primarily affecting the rod and cone photoreceptors and is characterized by night blindness, progressive visual field loss, and eventual loss of visual acuity3,4.

迄今为止，已鉴定出280多个基因负责IRD的发育，这些基因具有常染色体显性或隐性或X连锁遗传模式（RetNet，https://sph.uth.edu/retnet/上次访问时间为2024年2月）。色素性视网膜炎（RP，MIM#268000）是最常见的IRD，主要影响视杆细胞和视锥细胞感光细胞，其特征是夜盲症，进行性视野丧失和最终视力丧失3,4。

The molecular etiology of RP is complicated5,6, and mutations in at least 80 genes have been postulated as responsible for causing RP (RetNet).The RP1gene is associated with RP and encodes a microtubule-associated protein localized to connecting cilia of rod and cone photoreceptors. This protein is required for stability and organization of the disc membranes in the outer segment7,8,9.

RP的分子病因很复杂[5,6]，至少有80个基因的突变被认为是引起RP（RetNet）的原因。RP1基因与RP相关，并编码一种微管相关蛋白，该蛋白定位于杆状和锥形感光器的连接纤毛。这种蛋白质是外节段椎间盘膜稳定性和组织所必需的7,8,9。

RP1has been implicated in both autosomal dominant RP (adRP) and autosomal recessive RP (arRP), accounting for approximately 5.5% and 1% of cases, respectively10. More than 280 disease-causing mutations of RP1 listed in the Human Gene Mutation Database (HGMD, professional version 2023.4), and most are truncating variants.Recently, an insertion of the mobile element Alu in exon 4 of RP1was reported as a founder mutation in Japanese subjects with IRD11,12,13.

RP1与常染色体显性RP（adRP）和常染色体隐性RP（arRP）有关，分别约占病例的5.5%和1%。人类基因突变数据库（HGMD，专业版2023.4）中列出了280多种RP1致病突变，其中大多数是截短变体。最近，据报道，在日本IRD11,12,13受试者中，在RP1外显子4中插入了移动元件Alu，这是一种创始人突变。

The AluY insertion resulted in 328 additional nucleotides in ex.

AluY插入在ex中产生了328个额外的核苷酸。

Among the 1,072 samples, the AluY insertion was detected as heterozygous in six patients, all of which were found in the IRD group with genotype frequency of 0.0146 (6/411; 95% CI, 0.0067–0.0315) and allele frequency of 0.0073 (6/822; 95% CI, 0.0033–0.0158). An AluY insertion was not observed in non-IRD group (0/661) (Fig. 1).Fig.

在1072例样本中，6例患者检测到AluY插入为杂合子，均在IRD组中发现，基因型频率为0.0146（6/411；95%CI，0.0067-0.0315），等位基因频率为0.0073（6/822；95%CI，0.0033-0.0158）。在非IRD组（0/661）中未观察到AluY插入（图1）。图。

1Study population consisting of 1,072 subjects including 411 patients with inherited retinal diseases (IRDs group) and 661 with underlying genetic diseases other than eye diseases (non-IRD group) for screening of AluY insertion in exon 4 of RP1.Full size image.

1研究人群由1072名受试者组成，其中包括411名遗传性视网膜疾病患者（IRD组）和661名患有眼部疾病以外的潜在遗传疾病（非IRD组），用于筛查RP1外显子4中的AluY插入。全尺寸图像。

RP1 variant spectrum accompanying the AluY insertionThe AluY insertion in RP1 was found in six patients in the IRD group, five with RP and one with cone dystrophy. All patients were confirmed to have pathogenic variant in trans with the AluY insertion as follows: c.2398 A > G: p.(Lys800Ter) (N = 1), c.4196del: p.(Cys1399LeufsTer5) (N = 2), c.5797 C > T: p.(Arg1933Ter) (N = 1), and c.6181del: p.(Ile2061SerfsTer12) (N = 2).

伴随AluY插入的RP1变异谱在IRD组的6名患者中发现了RP1中的AluY插入，其中5名患有RP，1名患有锥体营养不良。。

The clinical characteristics and genetic data for these AluY insertion cases are summarized in Table 1.Table 1 Clinical characteristics and genotypes of six inherited retinal disease patients with AluY insertion.Full size tableDiagnostic pitfalls associated with AluY insertion in RP1.

表1总结了这些AluY插入病例的临床特征和遗传数据。表1六名AluY插入的遗传性视网膜疾病患者的临床特征和基因型。与RP1中AluY插入相关的全尺寸表诊断缺陷。

AluY allele dropout leading to false homozygosity for RP1 c.4196del variantIn two unrelated RP patients (study IDs: 41 and 89), the pathogenic variant c.4196del was detected along with the AluY insertion using targeted panel sequencing. In the subsequent Sanger validation, allele dropout (ADO) was suspected due to c.4196del variant homozygosity (Fig. 2A).

AluY等位基因缺失导致RP1 c.4196del变体的假纯合性在两名不相关的RP患者（研究ID:41和89）中，使用靶向面板测序检测到致病变体c.4196del以及AluY插入。在随后的Sanger验证中，由于c.4196del变异纯合性，怀疑等位基因缺失（ADO）（图2A）。

We performed repeated sequencing using redesigned primers for exon 4 to avoid the AluY insertion as shown in Fig. 2B and confirmed that dropout of the AluY allele causes a false-negative result, and dropout of the wild-type allele caused a heterozygous c.4196del variant to appear homozygous (Fig. 2B; Supplementary Table S1).

我们使用重新设计的外显子4引物进行了重复测序，以避免AluY插入，如图2B所示，并证实AluY等位基因的缺失会导致假阴性结果，而野生型等位基因的缺失会导致杂合的c.4196del变体看起来是纯合的（图2B；补充表S1）。

The ADO was presumed to be caused by expansion of the allele size due to an AluY insertion in the sample DNA, resulting in non-amplification of one of the two alleles.Fig. 2Error in detection of the c.4196del variant in RP1 due to AluY allele dropout (ADO) in two families with retinitis pigmentosa. (a) Homozygous deletion of the RP1 c.4196del variant in two unrelated RP patients.

据推测，ADO是由样品DNA中的AluY插入引起的等位基因大小扩大引起的，导致两个等位基因之一未扩增。图2在两个色素性视网膜炎家族中，由于AluY等位基因缺失（ADO），检测到RP1中c.4196del变体的错误。（a）两名无关的RP患者中RP1 c.4196del变体的纯合缺失。

(b) Resequencing with redesigned primers (P2-F and P2-R) avoiding the AluY insertion detected heterozygous deletion of the RP1 c.4196del variant in the two patients.Full size imageAllele phasing to discriminate mutant alleles for AluY and c.4196delPhase information is important for diagnosis of arRP caused by compound heterozygous mutations.

（b）用重新设计的引物（P2-F和P2-R）进行重测序，避免了AluY插入检测到两名患者中RP1 c.4196del变体的杂合缺失。全尺寸图像等位基因定相以区分突变等位基因的AluY和c.4196delPhase信息对于诊断由复合杂合突变引起的arRP很重要。

To resolve cis or trans ambiguities between mutated alleles for AluY and c.4196del genetic backgrounds, we designed one set of specific amplification reaction based on the specific primers and AS-PCR protocols (Fig. 3A and Supplementary Table S2). The specificity of the AS-PCR reaction involved a single 3’ mismatched nucleotide for c.4196del, which was sufficient to prevent extension.

为了解决AluY和c.4196del遗传背景突变等位基因之间的顺式或反式歧义，我们基于特异性引物和AS-PCR方案设计了一组特异性扩增反应（图3A和补充表S2）。AS-PCR反应的特异性涉及c.4196del的单个3'错配核苷酸，足以防止延伸。

We screened sequencing data of 1,072 individuals of Korean descent from the IRD and non-IRD groups using the Grep search program to detect the AluY insertion in exon 4 of RP1. The Grep program is an in silico method for detecting AluY insertion in RP1from FASTQ files through next-generation sequencing, based on the known sequence of the mutant junction14.

我们使用Grep搜索程序筛选了来自IRD和非IRD组的1072名韩国血统个体的测序数据，以检测RP1外显子4中的AluY插入。Grep程序是一种计算机方法，用于根据突变连接的已知序列14，通过下一代测序检测FASTQ文件中RP1中的AluY插入。

The Linux Grep command was used to search FASTQ files for the chimeric sequence of the 5’ junction between the reference sequence of exon 4 and the beginning of the AluY insertion in RP114. The chimeric sequences were forward, 5’-CCAAAGAAAACACggccgggcgcggt-3’; reverse, 5’-accgcgcccggccGTGTTTTCTTTGG-3’ (lowercase bases are the AluY sequence).

Linux Grep命令用于在FASTQ文件中搜索外显子4的参考序列与RP114中AluY插入开始之间的5'连接的嵌合序列。嵌合序列是正向的，5'-CCAAAGAAAACACGGCGGGCGCGGT-3'；反向，5'-ACCGCCCGGCCGTGTTTTCTTGG-3'（小写碱基是AluY序列）。

When AluY insertion was suspected in the screening results of the Grep search, soft-clipped sequences from the BAM file were visually inspected using the Integrative Genomics Viewer (IGV) to determine if the observed sequence was identical to that of the known AluY retrotransposon junction..

当在Grep搜索的筛选结果中怀疑AluY插入时，使用Integrative Genomics Viewer（IGV）目视检查BAM文件中的软剪切序列，以确定观察到的序列是否与已知的AluY逆转录转座子连接相同。。

AluY allele-specific PCR and gel electrophoresisWe performed AluY allele-specific PCR (AS-PCR) and gel electrophoresis to confirm the suspected AluY insertion in RP1 from the Grep search and IGV visual inspection. To target the AluY allele-specific sequence, we designed primers to amplify 239 bp, including 145 bp upstream and 83 bp downstream of target site duplication (sequence: 5’-AAAGAAAACAC-3’; Supplementary Table S5).

AluY等位基因特异性PCR和凝胶电泳我们进行了AluY等位基因特异性PCR（AS-PCR）和凝胶电泳，以通过Grep搜索和IGV目视检查确认RP1中可疑的AluY插入。为了靶向AluY等位基因特异性序列，我们设计了引物以扩增239 bp，包括靶位点重复上游145 bp和下游83 bp（序列：5'-AAAGAAAACAC-3'；补充表S5）。

Experimental condition for AS-PCR is described in detail in the Supplementary methods. The AluY insertion between c.4052 and c.4053 in exon 4 of RP1 was confirmed when a 328-bp insertion was identified on agarose gel electrophoresis of the tested sample. For the subsequent Sanger validation, PCR products were sequenced on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) using a BigDye Terminator Cycle sequencing kit (Applied Biosystems).

AS-PCR的实验条件在补充方法中有详细描述。当在测试样品的琼脂糖凝胶电泳上鉴定出328 bp的插入时，证实了RP1外显子4中c.4052和c.4053之间的AluY插入。为了进行随后的Sanger验证，使用BigDye终止子循环测序试剂盒（Applied Biosystems）在ABI 3730xl DNA分析仪（Applied Biosystems，Foster City，CA，USA）上对PCR产物进行测序。

Sequences were analyzed using Sequencher software (Gene Codes Corp., Ann Arbor, MI, USA) and were compared with the reference sequences for RP1 (NM_006269.1) and AluY (GenBank accession number JN391998.1). Gel images adhered to digital image and integrity policies and were obtained from samples processed in parellel from the same experiment.Long-read sequencingFor phase determination, long-read sequencing was performed by MinION flowcell (version 9.4.1) (Oxford Nanopore Technology).

使用Sequencher软件（Gene Codes Corp.，Ann Arbor，MI，USA）分析序列，并将其与RP1（NM\U 006269.1）和AluY（GenBank登录号JN391998.1）的参考序列进行比较。。长读测序对于相测定，长读测序由MinION flowcell（版本9.4.1）（Oxford Nanopore Technology）进行。

For cost-effectiveness of sequencing, last exon of RP1 in which Alu inserted was enriched by CRISPR-Cas9 system. CRISPR RNA (crRNA) was designed spanning 7,506 bp region by CHOPCHOP (https://chopchop.cbu.uib.no/) as follows; crRNA: 5’-ACCGCAATCTCAAGCAGAAG-3’ and 5’-GGTACTGTTACCCATCGAGA-3’. Experiments were done according to the protocol (version ENR_9084_v109_revD_04Dec2018).

为了提高测序的成本效益，CRISPR-Cas9系统富集了插入Alu的RP1的最后一个外显子。通过CHOPCHOP设计了跨越7506 bp区域的CRISPR RNA（crRNA）(https://chopchop.cbu.uib.no/)如下所示；crRNA:5'-ACCGCAATCAAGCAGAAG-3'和5'-GGTACTGTTACCATCGAGA-3'。根据协议（版本ENR\U 9084\U v109\U revD\U 04Dec2018）进行实验。

Base calling and alignment were conducted b.

b进行了碱基检出和比对。

Data availability

数据可用性

Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request.

根据合理的要求，通讯作者可以提供支持本手稿发现的数据。

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Download referencesFundingThis study was supported by a grant (No. 2021R1F1A1046537) of the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) and a grant (No. SMO1220671) from Samsung Medical Center.Author informationAuthors and AffiliationsDepartment of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, KoreaMi-Ae Jang, Jong Kwon Lee, Young-gon Kim, Jong-Won Kim, Youn-Ji Hong & Ja-Hyun JangClinical Genome Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, KoreaJong-Ho ParkDepartment of Ophthalmology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, KoreaSungsoon Hwang & Sang Jin KimAuthorsMi-Ae JangView author publicationsYou can also search for this author in.

下载参考文献资助这项研究得到了韩国政府（MSIT）资助的韩国国家研究基金会（NRF）的资助（编号2021R1F1A1046537）和三星医疗中心的资助（编号SMO1220671）。作者信息作者和附属机构首尔成均馆大学医学院三星医学中心实验医学与遗传学系，KoreaMi Ae Jang，Jong Kwon Lee，Young gon Kim，Jong Won Kim，Youn Ji Hong＆Ja Hyun Jang临床基因组中心，成均馆大学医学院三星医学中心，首尔，韩国成均馆大学医学院三星医学中心眼科系，首尔，KoreaSungsoon Hwang＆Sang Jin Kimouthorsmi Ae JangView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsJ-HJ planned the study. M-AJ and JKL performed data curation and investigation. M-AJ performed the data analysis, data visualization, and writing-original draft. J-HP, Y-JH, SH, Y-GK, and J-WK involved in sample collection and investigation. SJK and J-HJ performed supervision, writing-review and editing.

PubMed Google ScholarContributionsJ HJ计划了这项研究。。M-AJ进行了数据分析，数据可视化和撰写原始草案。J-HP，Y-JH，SH，Y-GK和J-WK参与样本收集和调查。SJK和J-HJ进行监督，撰写评论和编辑。

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Reprints and permissionsAbout this articleCite this articleJang, MA., Lee, J.K., Park, JH. et al. Identification of diagnostic challenges in RP1 Alu insertion and strategies for overcoming them.

转载和许可本文引用本文Jang，MA。，Lee，J.K.，Park，JH。等人。确定RP1 Alu插入中的诊断挑战以及克服这些挑战的策略。

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Keywords

关键词

Alu elementsAllele frequency

铝元素等频率

RP1

RP1型

RetinaRetinitis pigmentosaVision disorders

视网膜视网膜色素变性视力障碍

Subjects

主题