单分子实时测序技术在1例21-羟化酶缺陷症患儿基因检测中的临床应用

doi:10.6040/j.issn.1671-7554.0.2023.1017

摘要/Abstract

摘要： 目的应用单分子实时测序(single molecular real-time sequencing, SMRT)技术明确1例21-羟化酶缺陷症患儿的分子病因,并探讨SMRT用于临床基因检测的可行性。方法应用SMRT技术对先证者先天性肾上腺皮质增生症候选基因进行长读长测序,检测结果与多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)和Sanger测序检测的家系结果进行比较。结果 SMRT结果提示,先证者CYP21A2基因存在3个致病变异,先证者的一条染色体上串联排列两个重复的基因拷贝,分别为包含c.955C>T变异的CYP21A2基因和包含c.1069C>T变异的CYP21A2/CYP21A1P嵌合基因;另一染色体上的CYP21A2基因缺失。检测结果与家系MLPA+Sanger测序的结果相同,并明确了CYP21A2/CYP21A1P嵌合基因的排列方式。结论 SMRT技术能够明确基因拷贝数变异、结构变异及嵌合基因的排列方式,可为21-羟化酶缺陷症遗传学诊断和携带者筛查提供更有价值的信息,具有较大的临床应用前景。

关键词: 单分子实时测序, 长读长测序, CYP21A2基因, 嵌合基因, 基因重复

Abstract: Objective To determine the molecular etiology in a child with 21-hydroxylase deficiency using single molecular real-time sequencing(SMRT), and explore its clinical application for gene detection. Methods SMRT technology was applied to perform long-read sequencing on the candidate gene for congenital adrenal hyperplasia in the proband, and the results were compared to those obtained from multiplex ligation-dependent probe amplification(MLPA)and Sanger sequencing in the family. Results The SMRT results revealed three pathogenic variants in the probands CYP21A2 gene, including two tandemly arranged gene copies on one chromosome(one CYP21A2 copy with c.955C>T mutation and the other CYP21A2/CYP21A1P chimeric copy with a c.1069C>T mutation), and a deletion of the CYP21A2 gene on the other chromosome. These variants were consistent with the results obtained by MLPA+Sanger sequencing in the family, and the arrangement of the CYP21A2/CYP21A1P chimeric gene was clarified. Conclusion SMRT technology can identify gene copy number variations, structure variations, and chimeric genes, providing more valuable information for genetic diagnosis and carrier screening of 21-hydroxylase deficiency.

Key words: Single molecular real-time sequencing, Long-read sequencing, CYP21A2 gene, Chimeric gene, Gene duplication

中图分类号:

R586.2

赵炜,王芳,李加山,梁思颖,苗艳,姜楠,李朔. 单分子实时测序技术在1例21-羟化酶缺陷症患儿基因检测中的临床应用[J]. 山东大学学报 (医学版), 2024, 62(3): 70-76.

ZHAO Wei, WANG Fang, LI Jiashan, LIANG Siying, MIAO Yan, JIANG Nan, LI Shuo. Clinical application of single molecule real-time sequencing technology in gene detection for a child with 21-hydroxylase deficiency[J]. Journal of Shandong University (Health Sciences), 2024, 62(3): 70-76.

参考文献

[1] Merke D, Kabbani M. Congenital adrenal hyperplasia[J]. Paediatr Drugs, 2001, 3(8): 599-611.
[2] Speiser PW, Arlt W, Auchus RJ, et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an endocrine society clinical practice guideline[J]. J Clin Endocrinol Metab, 2018, 103(11): 4043-4088.
[3] Concolino P, Costella A. Congenital adrenal hyperplasia(CAH)due to 21-hydroxylase deficiency: a comprehensive focus on 233 pathogenic variants of CYP21A2 gene[J]. Mol Diagn Ther, 2018, 22(3): 261-280.
[4] 孙昱, 邬玲仟, 叶蕾, 等. 21羟化酶缺陷导致的先天性肾上腺皮质增生症的实验室诊断共识[J]. 中华医学遗传学杂志, 2023, 40(7): 769-780. SUN Yu, WU Lingqian, YE Lei, et al. Consensus on the laboratory diagnosis of congenital adrenal hyperplasia due to 21 hydroxylase deficiency[J]. Chinese Journal of Medical Genetics, 2023, 40(7): 769-780.
[5] 马定远, 孙云, 陈玉林, 等. 21-羟化酶缺陷症基因诊断方法的建立及应用[J]. 中华医学遗传学杂志, 2013, 30(1): 49-54. MA Dingyuan, SUN Yun, CHEN Yulin, et al. Development and application of a method for molecular diagnosis of 21-hydroxylase deficiency[J]. Chinese Journal of Medical Genetics, 2013, 30(1): 49-54.
[6] 高寅洁, 于冰青, 卢琳, 等. 多重连接探针扩增技术联合Sanger测序对21-羟化酶缺陷症的诊断价值[J]. 中华医学杂志, 2019, 99(6): 432-437. GAO Yinjie, YU Bingqing, LU Lin, et al. Diagnostic value of multiplex ligation dependent probe amplification combined with Sanger sequencing in 21-hydroxylase deficiency[J]. National Medical Journal of China, 2019, 99(6): 432-437.
[7] 中华医学会儿科学分会内分泌遗传代谢病学组. 先天性肾上腺皮质增生症21-羟化酶缺陷诊治共识[J]. 中华儿科杂志, 2016, 54(8): 569-576.
[8] 夏艳洁, 梅世月, 胡爽, 等. 18个21-羟化酶缺陷症高危家系的基因筛查和产前诊断[J]. 中华医学遗传学杂志, 2019, 36(2): 103-107. XIA Yanjie, MEI Shiyue, HU Shuang, et al. Genetic screening and prenatal diagnosis in 18 high-risk families with 21-hydroxylase deficiency[J]. Chinese Journal of Medical Genetics, 2019, 36(2): 103-107.
[9] Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[J]. Genet Med, 2015, 17(5): 405-424.
[10] White PC, New MI, Dupont B. Structure of human steroid 21-hydroxylase genes[J]. Proc Natl Acad Sci U S A, 1986, 83(14): 5111-5115.
[11] Blanchong CA, Zhou B, Rupert KL, et al. Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX(RCCX)modules in caucasians. The load of RCCX genetic diversity on major histocompatibility complex-associated disease[J]. J Exp Med, 2000, 191(12): 2183-2196.
[12] Claahsen-van der Grinten HL, Speiser PW, Ahmed SF, et al. Congenital adrenal hyperplasia-current insights in pathophysiology, diagnostics, and management[J]. Endocr Rev, 2022, 43(1): 91-159.
[13] Chen WY, Xu Z, Sullivan A, et al. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency[J]. Clin Chem, 2012, 58(2): 421-430.
[14] 常晨凯. 21-羟化酶缺陷症基因型与表型相关性研究[D]. 北京: 北京大学, 2021.
[15] Fanis P, Skordis N, Toumba M, et al. The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes[J]. Front Endocrinol, 2023, 14: 1156616. doi:10.3389/fendo.2023.1156616.
[16] Kleinle S, Lang R, Fischer GF, et al. Duplications of the functional CYP21A2 gene are primarily restricted to Q318X alleles: evidence for a founder effect[J]. J Clin Endocrinol Metab, 2009, 94(10): 3954-3958.
[17] Carrozza C, Foca L, de Paolis E, et al. Genes and Pseudogenes: complexity of the RCCX locus and disease[J]. Front Endocrinol, 2021, 12: 709758. doi:10.3389/fendo.2021.709758.
[18] Rhoads A, Au KF. PacBio sequencing and its applications[J]. Genomics Proteomics Bioinformatics, 2015, 13(5): 278-289.
[19] Liu YD, Chen MM, Liu J, et al. Comprehensive analysis of congenital adrenal hyperplasia using long-read sequencing[J]. Clin Chem, 2022, 68(7): 927-939.
[20] Adachi E, Nakagawa R, Tsuji-Hosokawa A, et al. A minION-based long-read sequencing application with one-step PCR for the genetic diagnosis of 21-hydroxylase deficiency[J]. J Clin Endocrinol Metab, 2024, 109(3): 750-760.
[21] Arriba M, Ezquieta B. Molecular diagnosis of steroid 21-hydroxylase deficiency: a practical approach[J]. Front Endocrinol, 2022, 13: 834549. doi:10.3389/fendo.2022.834549.
[22] Lee HH, Niu DM, Lin RW, et al. Structural analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency[J]. J Hum Genet, 2002, 47(10): 517-522.
[23] Lao QZ, Burkardt DD, Kollender S, et al. Congenital adrenal hyperplasia due to two rare CYP21A2 variant alleles, including a novel attenuated CYP21A1P/CYP21A2 chimera[J]. Mol Genet Genomic Med, 2023, 11(7): e2195. doi:10.1002/mgg3.2195.
[24] Menabò S, Balsamo A, Baldazzi L, et al. A sequence variation in 3'UTR of CYP21A2 gene correlates with a mild form of congenital adrenal hyperplasia[J]. J Endocrinol Invest, 2012, 35(3): 298-305.
[25] Paragliola RM, Perrucci A, Foca L, et al. Prevalence of CAH-X syndrome in Italian patients with congenital adrenal hyperplasia(CAH)due to 21-hydroxylase deficiency[J]. J Clin Med, 2022, 11(13): 3818. doi:10.3390/jcm11133818.
[26] Kim JH, Kim GH, Yoo HW, et al. Molecular basis and genetic testing strategies for diagnosing 21-hydroxylase deficiency, including CAH-X syndrome[J]. Ann Pediatr Endocrinol Metab, 2023, 28(2): 77-86.
[27] Fanis P, Skordis N, Phylactou LA, et al. Salt-wasting congenital adrenal hyperplasia phenotype as a result of the TNXA/TNXB chimera 1(CAH-X CH-1)and the pathogenic IVS2-13A/C>G in CYP21A2 gene[J]. Hormones, 2023, 22(1): 71-77.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed