脆性X综合征遗传学诊断方法研究进展

doi:10.3969/j.issn.1673-8640.2024.02.002

摘要/Abstract

摘要：

脆性X综合征(FXS)是导致智力障碍和发育障碍的主要单基因病之一，呈X连锁不完全显性遗传。FXS的病因是FMR1基因内(CGG)n重复序列的不稳定扩展及其上游CpG岛的异常甲基化，进而导致脆性X智力低下蛋白(FMRP)减少或缺乏，FMRP的水平直接关系到临床表型的严重程度。临床表现和基因检测是诊断FXS的主要依据。然而，FMR1基因分子结构和遗传模式的特殊性使得FXS的分子诊断和遗传咨询面临挑战。因此，如何简便而准确地进行FMR1基因检测一直是临床关注的焦点。文章针对FXS遗传学诊断方法的研究进展进行综述，旨在促进FXS的规范诊断，为临床提供帮助。

关键词: FMR1基因, (CGG)n重复, 脆性X综合征, 基因诊断

Abstract:

Fragile X syndrome(FXS) is one of the main monogenic diseases that cause intellectual and developmental disorders，and it shows an X-linked incomplete dominant inheritance. The etiology of FXS is unstable extension of (CGG)n repeat within FMR1 gene and abnormal methylation of its upstream CpG island，which would lead to decreasing or deficiency of fragile X mental retardation protein(FMRP). The level of FMRP is directly related to the severity of clinical phenotype. Clinical manifestation and genetic testing are the main diagnostic criteria for FXS. However，the unique molecular structure and genetic pattern of FMR1 gene pose challenges to the molecular diagnosis and genetic counseling of FXS. Therefore，how to detect FMR1 gene conveniently and accurately has always been a focus of attention. This review focuses on the research progress of FXS genetic diagnostic methods，aiming to promote the standardized diagnosis of FXS and provide assistance for clinic.

Key words: FMR1 gene, (CGG)n repeat, Fragile X syndrome, Genetic diagnosis

中图分类号:

R446.7

蒋祝, 谭建新, 谭娟, 罗春玉, 许争峰. 脆性X综合征遗传学诊断方法研究进展[J]. 检验医学, 2024, 39(2): 107-113.

JIANG Zhu, TAN Jianxin, TAN Juan, LUO Chunyu, XU Zhengfeng. Research progress in genetic diagnostic methods of fragile X syndrome[J]. Laboratory Medicine, 2024, 39(2): 107-113.

图/表 3

参考文献 47

[1]	CIOBANU C G, NUC I, POPESCU R, et al. Narrative review:update on the molecular diagnosis of fragile X syndrome[J]. Int J Mol Sci, 2023, 24(11):9206. DOI URL
[2]	SITZMANN A F, HAGELSTROM R T, TASSONE F, et al. Rare FMR1 gene mutations causing fragile X syndrome:a review[J]. Am J Med Genet A, 2018, 176(1):11-18. DOI URL
[3]	ALLEN E G, CHAREN K, HIPP H S, et al. Refining the risk for fragile X-associated primary ovarian insufficiency(FXPOI)by FMR1 CGG repeat size[J]. Genet Med, 2021, 23(9):1648-1655. DOI URL
[4]	中国医师协会医学遗传医师分会临床遗传学组, 中华医学会医学遗传学分会临床遗传学组, 中华预防医学会出生缺陷预防与控制专业委员会遗传病防控学组, 等. 脆性X综合征的临床实践指南[J]. 中华医学遗传学杂志, 2022, 39(11):1181-1186.
[5]	TASSONE F, ADAMS J, BERRY-KRAVIS E M, et al. CGG repeat length correlates with age of onset of motor signs of the fragile X-associated tremor/ataxia syndrome(FXTAS)[J]. Am J Med Genet B Neuropsychiatr Genet, 2007, 144B(4):566-569. DOI URL
[6]	SALCEDO-ARELLANO M J, HAGERMAN R J, MARTÍNEZ-CERDEÑO V. Fragile X syndrome:clinical presentation,pathology and treatment[J]. Gac Med Mex, 2020, 156(1):60-66.
[7]	MENG L, KAUFMANN W E, FRYE R E, et al. The association between mosaicism type and cognitive and behavioral functioning among males with fragile X syndrome[J]. Am J Med Genet A, 2022, 188(3):858-866. DOI URL
[8]	NOLIN S L, GLICKSMAN A, HOUCK G E, et al. Mosaicism in fragile X affected males[J]. Am J Med Genet, 1994, 51(4):509-512. DOI URL
[9]	NOLIN S L, BROWN W T, GLICKSMAN A, et al. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles[J]. Am J Hum Genet, 2003, 72(2):454-464. PMID
[10]	YRIGOLLEN C M, DURBIN-JOHNSON B, GANE L, et al. AGG interruptions within the maternal FMR1 gene reduce the risk of offspring with fragile X syndrome[J]. Genet Med, 2012, 14(8):729-736.
[11]	MONAGHAN K G, LYON E, SPECTOR E B, et al. ACMG standards and guidelines for fragile X testing:a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics[J]. Genet Med, 2013, 15(7):575-586.
[12]	HIRST M C, KNIGHT S J, BELL M V, et al. The fragile X syndrome[J]. Clin Sci(Lond), 1992, 83(3):255-264. DOI URL
[13]	JACKY P B, AHUJA Y R, ANYANE-YEBOA K, et al. Guidelines for the preparation and analysis of the fragile X chromosome in lymphocytes[J]. Am J Med Genet, 1991, 38(2-3):400-403. DOI URL
[14]	GOLD B, RADU D, BALANKO A, et al. Diagnosis of fragile X syndrome by southern blot hybridization using a chemiluminescent probe:a laboratory protocol[J]. Mol Diagn, 2000, 5(3):169-178. DOI
[15]	ZHOU Y, LUM J M, YEO G H, et al. Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis[J]. Clin Chem, 2006, 52(8):1492-1500. DOI PMID
[16]	BERRY-KRAVIS E, ZHOU L, JACKSON J, et al. Diagnostic profile of the amplidex fragile X Dx and carrier screen kit for diagnosis and screening of fragile X syndrome and other FMR1-related disorders[J]. Expert Rev Mol Diagn, 2021, 21(3):255-267. DOI URL
[17]	ADAM M P, FELDMAN J, MIRZAA G M, et al. FMR1 disorders[EB/OL].(1998-06-16)[2023-10-01]. https://www.ncbi.nlm.nih.gov/books/NBK1384/#_ncbi_dlg_citbx_NBK1384.
[18]	CHEN L, HADD A, SAH S, et al. High-resolution methylation polymerase chain reaction for fragile X analysis:evidence for novel FMR1 methylation patterns undetected in southern blot analyses[J]. Genet Med, 2011, 13(6):528-538.
[19]	KHODADADI E, FAHMIDEH L, KHODADADI E, et al. Current advances in DNA methylation analysis methods[J]. Biomed Res Int, 2021, 2021:8827516.
[20]	FILIPOVIC-SADIC S, SAH S, CHEN L, et al. A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome[J]. Clin Chem, 2010, 56(3):399-408. DOI URL
[21]	MUTHUSWAMY S, DEAN D D, AGARWAL S. Molecular screening of intellectually disabled patients and those with premature ovarian failure for CGG repeat expansion at FMR1 locus:implication of combined triplet repeat primed polymerase chain reaction and methylation-specific polymerase chain reaction analysis[J]. Neurol India, 2016, 64(6):1175-1179. DOI URL
[22]	XI H, XIE W, CHEN J, et al. Implementation of fragile X syndrome carrier screening during prenatal diagnosis:a pilot study at a single center[J]. Mol Genet Genomic Med, 2021, 9(7):e1711. DOI URL
[23]	GUO Q, CHANG Y Y, HUANG C H, et al. Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations[J]. J Genet Genomics, 2021, 48(12):1104-1110. DOI PMID
[24]	GU H, KIM M J, YANG D, et al. Accuracy and performance evaluation of triplet repeat primed PCR as an alternative to conventional diagnostic methods for fragile X syndrome[J]. Ann Lab Med, 2021, 41(4):394-400. DOI PMID
[25]	ZHANG J Y, WU D W, YANG R L, et al. FMR1 allele frequencies in 51 000 newborns:a large-scale population study in China[J]. World J Pediatr, 2021, 17(6):653-658. DOI
[26]	曹琴英, 穆卫红, 孙东兰, 等. 孕妇早、中期开展fmr1基因突变筛查的意义及病例分析[J]. 中华医学遗传学杂志, 2021, 38(5):450-453.
[27]	MEI L, HU C, LI D, et al. The incidence and clinical characteristics of fragile X syndrome in China[J]. Front Pediatr, 2023, 11:1064104. DOI URL
[28]	National Fragile X Foundation. Genetic testing for fragile X syndrome and associated disorders[EB/OL]. https://fragilex.org/understanding-fragile-x/fragile-x-101/testing-diagnosis/.
[29]	WELLS R D. Mutation spectra in fragile X syndrome induced by deletions of CGG*CCG repeats[J]. J Biol Chem, 2009, 284(12):7407-7411. DOI PMID
[30]	HEGDE M R, CHONG B, FAWKNER M, et al. Microdeletion in the FMR-1 gene:an apparent null allele using routine clinical PCR amplification[J]. J Med Genet, 2001, 38(9):624-629. DOI URL
[31]	CARROLL R, SHAW M, ARVIO M, et al. Two novel intragenic variants in the FMR1 gene in patients with suspect clinical diagnosis of fragile X syndrome and no CGG repeat expansion[J]. Eur J Med Genet, 2020, 63(10):104010. DOI URL
[32]	GROSSO V, MARCOLUNGO L, MAESTRI S, et al. Characterization of FMR1 repeat expansion and intragenic variants by indirect sequence capture[J]. Front Genet, 2021, 12:743230. DOI URL
[33]	LUGENBEEL K A, PEIER A M, CARSON N L, et al. Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome[J]. Nat Genet, 1995, 10(4):483-485. PMID
[34]	GRØNSKOV K, BRØNDUM-NIELSEN K, DEDIC A, et al. A nonsense mutation in FMR1 causing fragile X syndrome[J]. Eur J Hum Genet, 2011, 19(4):489-491. DOI
[35]	QUARTIER A, POQUET H, GILBERT-DUSSARDIER B, et al. Intragenic FMR1 disease-causing variants:a significant mutational mechanism leading to fragile-X syndrome[J]. Eur J Hum Genet, 2017, 25(4):423-431. DOI
[36]	LINDER B, PLÖTTNER O, KROISS M, et al. Tdrd3 is a novel stress granule-associated protein interacting with the fragile-X syndrome protein FMRP[J]. Hum Mol Genet, 2008, 17(20):3236-3246. DOI PMID
[37]	OKRAY Z, DE ESCH C E, VAN ESCH H, et al. A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function[J]. EMBO Mol Med, 2015, 7(4):423-437. DOI PMID
[38]	LIANG Q, LIU Y, LIU Y, et al. Comprehensive analysis of fragile X syndrome:full characterization of the FMR1 locus by long-read sequencing[J]. Clin Chem, 2022, 68(12):1529-1540. DOI URL
[39]	JIRAANONT P, SWEHA S R, ALOLABY R R, et al. Clinical and molecular correlates in fragile X premutation females[J]. E Neurological Sci, 2017, 7:49-56.
[40]	REFEAT M M, EL SAIED M M, ABDEL RAOUF E R. Diagnostic value of molecular approach in screening for fragile X premutation cases[J]. Ir J Med Sci, 2023, 192(5):2265-2272. DOI
[41]	PHOADS A, AU K F. PacBio sequencing and its applications[J]. Genomics Proteomics Bioinformatics, 2015, 13(5):278-289. DOI URL
[42]	CONLIN L K, AREF-ESHGHI E, MCELDREW D A, et al. Long-read sequencing for molecular diagnostics in constitutional genetic disorders[J]. Hum Mutat, 2022, 43(11):1531-1544. DOI PMID
[43]	WENGER A M, PELUSO P, ROWELL W J, et al. Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome[J]. Nat Biotechnol, 2019, 37(10):1155-1162. DOI PMID
[44]	WANG Y, ZHAO Y, BOLLAS A, et al. Nanopore sequencing technology,bioinformatics and applications[J]. Nat Biotechnol, 2021, 39(11):1348-1365. DOI
[45]	STEVANOVSKI I, CHINTALAPHANI S R, GAMAARACHCHI H, et al. Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing[J]. Sci Adv, 2022, 8(9):eabm5386. DOI URL
[46]	ERDMANN H, SCHÖBERL F, GIURGIU M, et al. Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing[J]. Brain, 2023, 146(5):1831-1843. DOI URL
[47]	KONO N, ARAKAWA K. Nanopore sequencing:review of potential applications in functional genomics[J]. Dev Growth Differ, 2019, 61(5):316-326. DOI URL

变异	变异类型	临床意义	参考文献
c.52-1_52delinsTA(p.Ala18Leufs*4；p.Ala18_Glu66del)	剪接位点变异	致病	[33]
c.80C>A(p.Ser27*)	无义变异	致病	[34]
c.373delA(p.Thr125Leufs*35)	移码变异	致病	[33]
c.420-8A>G(p.Met140Ilefs*3)	移码变异	致病	[35]
c.881-1G>T	剪接位点变异	致病	[31]
c.911T>A(p.Ile304Asn)	错义变异	致病	[36]
c.990+1G>A(p.Lys295Asnfs*11)	剪接位点变异	致病	[35]
c.1021-1028delinsTATTGG(p.Asn341Tyrfs*7)	移码变异	致病	[31]
c.1611insG(p.Gly538Argfs24)	移码变异	致病	[37]
c.(1737+1_1738-1)_(1_?)del(p.Ile580fs9)	移码变异	致病	[35]
c.879A>C	剪接位点变异	致病	[38]

变异	变异类型	临床意义	参考文献
c.52-1_52delinsTA(p.Ala18Leufs*4；p.Ala18_Glu66del)	剪接位点变异	致病	[33]
c.80C>A(p.Ser27*)	无义变异	致病	[34]
c.373delA(p.Thr125Leufs*35)	移码变异	致病	[33]
c.420-8A>G(p.Met140Ilefs*3)	移码变异	致病	[35]
c.881-1G>T	剪接位点变异	致病	[31]
c.911T>A(p.Ile304Asn)	错义变异	致病	[36]
c.990+1G>A(p.Lys295Asnfs*11)	剪接位点变异	致病	[35]
c.1021-1028delinsTATTGG(p.Asn341Tyrfs*7)	移码变异	致病	[31]
c.1611insG(p.Gly538Argfs24)	移码变异	致病	[37]
c.(1737+1_1738-1)_(1_?)del(p.Ile580fs9)	移码变异	致病	[35]
c.879A>C	剪接位点变异	致病	[38]

项目	(CGG)n重复数识别能力		女性杂合子/纯合子判断	评估甲基化状态	分辨低比例嵌合体	检测AGG嵌入数	检测前突变	检测SNV/InDel
模型分析	不能		分辨率低	不能	分辨率低	不能	不能	不能
DNA印迹杂交法	偏低，无法精准确定CGG的重复次数		分辨率低	能	分辨率低	不能	能	不能
MS-PCR	偏低		不能	能	不能	不能	不能	不能
TP-PCR	中等，对于较大重复数不能进行精确定量分析，存在一定错判全突变型和前突变型的概率		分辨率高	不能	分辨率较高	可识别AGG插入有限(不能识别女性杂合和嵌合等位基因中的AGG)	能	不能
RT-PCR	不能		不能	不能	不能	不能	不能	不能
NGS	不能		不能	不能	不能	不能	不能	能
TGS	高		分辨率高	能	分辨率高	可识别AGG插入数量和位置(包括杂合和嵌合等位基因中的AGG)	能	能
项目	检测mRNA	样本需要量	检测时间	检测灵敏度	检测特异性	操作复杂程度
模型分析	不能	高(外周血细胞)	7 d	低	低	复杂
DNA印迹杂交法	不能	高(5~10 μg)	3 d	低(嵌合体影响灵敏度)	低	复杂
MS-PCR	不能	较高(2 μg)	>1 d	高	高	简单
TP-PCR	不能	低(5~100 ng)	<24 h	高	高	简单
RT-PCR	能	低(5~100 ng)	<24 h	高	高	简单
NGS	不能	中(500 ng)	>24 h	高	高	相对复杂
TGS	不能	中(500 ng)	12~24 h	高	高	相对复杂

项目	(CGG)n重复数识别能力		女性杂合子/纯合子判断	评估甲基化状态	分辨低比例嵌合体	检测AGG嵌入数	检测前突变	检测SNV/InDel
模型分析	不能		分辨率低	不能	分辨率低	不能	不能	不能
DNA印迹杂交法	偏低，无法精准确定CGG的重复次数		分辨率低	能	分辨率低	不能	能	不能
MS-PCR	偏低		不能	能	不能	不能	不能	不能
TP-PCR	中等，对于较大重复数不能进行精确定量分析，存在一定错判全突变型和前突变型的概率		分辨率高	不能	分辨率较高	可识别AGG插入有限(不能识别女性杂合和嵌合等位基因中的AGG)	能	不能
RT-PCR	不能		不能	不能	不能	不能	不能	不能
NGS	不能		不能	不能	不能	不能	不能	能
TGS	高		分辨率高	能	分辨率高	可识别AGG插入数量和位置(包括杂合和嵌合等位基因中的AGG)	能	能
项目	检测mRNA	样本需要量	检测时间	检测灵敏度	检测特异性	操作复杂程度
模型分析	不能	高(外周血细胞)	7 d	低	低	复杂
DNA印迹杂交法	不能	高(5~10 μg)	3 d	低(嵌合体影响灵敏度)	低	复杂
MS-PCR	不能	较高(2 μg)	>1 d	高	高	简单
TP-PCR	不能	低(5~100 ng)	<24 h	高	高	简单
RT-PCR	能	低(5~100 ng)	<24 h	高	高	简单
NGS	不能	中(500 ng)	>24 h	高	高	相对复杂
TGS	不能	中(500 ng)	12~24 h	高	高	相对复杂