目的

探讨基于长片段-聚合酶链反应（LR-PCR）与长读长测序（LRS）的三代测序技术结合的脆性X 染色体综合征（FXS）综合分析方法（CAFXS），应用于脆性X 智力低下1（FMR1）基因检测的临床应用。

方法

选择2021年7月至2023年7月于南京市妇幼保健院接受扩展性携带者筛查 （ECS）、遗传咨询门诊就诊的FXS患者/FXS前突变携带者及其家系成员共计33例受试者为研究对象。对这33例受试者均采用CAFXS进行FMR1 基因的CGG 重复数、AGG 插入、1号外显子单核苷酸变异（SNV）、小片段插入/缺失（InDel）和微缺失检测。同时采用三核苷酸重复引物-PCR（TP-PCR）结合毛细管电泳（CE）检测CGG 重复数，并对比上述2种方法的检测结果。本研究遵循的程序经南京市妇幼保健院伦理委员会批准（2024KY-152）。所有受试者由本人或者其监护人签署本研究知情同意书。

结果

①CAFXS与TP-PCR/CE 对33例受试者的FMR1 基因分型结果完全一致， 其中10例为FMR1基因全突变型、9例为前突变型、7例为中间型和7例为正常型。②33例受试者中，共有73个AGG 插入，涉及36种不同AGG 插入模式，其中频率最高的AGG 插入模式为9A9A9（24.6%）。③33例受试者中，CAFXS检测到9例（27.3%）FMR1基因CGG 重复嵌合型，包括前突变/全突变型嵌合（6例）、全突变型嵌合（2例）和正常型嵌合（1例）。④33例受试者均未检测到FMR1 基因1号外显子罕见SNV、In Del和微缺失致病变异。

结论

CAFXS可全面、准确地进行FMR1 基因分析，可为临床提供更多遗传信息。

Objective

To explore the detection performance of comprehensive analysis of fragile X syrdrome （CAFXS）based on long fragment PCR （LR-PCR）combined with long read sequencing（LRS）in the detection of fragile X mental retardation 1（FMR1）gene.

Methods

A total of 33 participants with FXS/premutation carriers and their family members who underwent expanded carrier screening or visited the genetic counseling clinic at Nanjing Women and Children's Healthcare Hospital from July 2021 to July 2023 were selected into this study.CAFXS was adopted to detect CGG repeats，AGG insertions of FMR1 gene，and single nucleotide variation （SNV），insertions/deletions（InDels），microdeletions within exon 1 of FMR1 gene.At the same time，trinucleotide repeat primer PCR （TP-PCR）combined with capillary electrophoresis（CE）was used to detect the CGG repeats，and were compared the detection results with CAFXS were compared.This study was approved by the Ethics Committee of Women and Children's Healthcare Hospital （2024KY-152）.Informed consents were obtained from participants or their guardians.

Results

①The results of CAFXS and TP-PCR/CE for detecting FMR1 genotype were consistent.Among the 33 participants，there were 10 cases of full mutation，9 cases of pre-mutation，7 cases of intermediate type and 7 cases of normal type.②Among the 33 participants，CAFXS detected 73 AGG insertions in 33 subjects，involving 36 AGG insertion patterns，among which the highest frequency was 9A9A9（24.6%）.③Among the 33 participants，CAFXS detected 9 cases （27.3%）of CGG repeats mosaicism，including pre-mutation/full-mutation mosaicism（6 cases），full-mutation mosaicism（2 cases）and normal mosaicism（1 case）.④No rare SNV，InDel and microdeletions within exon 1 of FMR1 gene were detected in 33 subjects.

Conclusions

CAFXS could analyze FMR1 gene comprehensively and accurately，and could provide more genetic information for clinical practice.