胚胎植入前遗传学检测在阻断常染色体隐性多囊肾病家系多囊肾/多囊肝病变1基因新突变遗传的应用

doi:10.3969/j.issn.1006-5725.2024.07.022

实用医学杂志 ›› 2024, Vol. 40 ›› Issue (7): 1006-1010.doi: 10.3969/j.issn.1006-5725.2024.07.022

胚胎植入前遗传学检测在阻断常染色体隐性多囊肾病家系多囊肾/多囊肝病变1基因新突变遗传的应用

王凝^1,^2,³,郝燕^1,^2,³(),陈大蔚^1,^4,^5,⁶,章志国^1,^4,^5,⁶,匡丹^1,^4,⁵,张清^1,^4,⁵,尹奕琪^1,^4,⁵,魏兆莲^1,^4,^5,⁶,周平^1,^4,^5,⁶,曹云霞^1,^2,³

^1.安徽医科大学第一附属医院妇产科（合肥 230000 ）
^2.国家卫生健康委配子及生殖道异常研究重点实验室（合肥 230000 ）
^3.出生人口健康教育部重点实验室（合肥 230000 ）
^4.生殖健康与遗传安徽省重点实验室（合肥 230000 ）
^5.安徽省生命资源保存与人工器官工程技术研究中心（合肥 230000 ）
^6.安徽省转化医学研究院（合肥 230000 ）

收稿日期:2023-10-30 出版日期:2024-04-10 发布日期:2024-04-08
通讯作者: 郝燕 E-mail:yan1987215@126.com
基金资助:
国家自然科学基金项目(82001631);安徽省科技重大专项(202003a07020012);安医大附院博士人才基金项目(1465);安徽医科大学校科研基金项目(2023xkj135)

Application research of PGT in blocking the inheritance of novel mutations in the PKHD1 gene in autosomal recessive polycystic kidney disease pedigrees

Ning WANG^1,^2,³,Yan HAO^1,^2,³(),Dawei CHEN^1,^4,^5,⁶,Zhiguo ZHANG^1,^4,^5,⁶,Dan KUANG^1,^4,⁵,Qing ZHANG^1,^4,⁵,Yiqi YING^1,^4,⁵,Zhaolian WEI^1,^4,^5,⁶,Ping ZHOU^1,^4,^5,⁶,Yunxia CAO^1,^2,³

^1.Department of Obstetrics and Gynecology，the First Affiliated Hospital of Anhui Medical University，Hefei 230000，China
^2.Key Laboratory of Study on Abnormal Gametes and Reproductive Tract，National Health Commission，Hefei 230000，China
^3.Key Laboratory of Population Health Across Life Cycle，Ministry of Education of the People′s Republic of China，Hefei 230000，China

Received:2023-10-30 Online:2024-04-10 Published:2024-04-08
Contact: Yan HAO E-mail:yan1987215@126.com

摘要/Abstract

摘要：

目的探讨基于二代测序（NGS）技术的单核苷酸多态性（SNP）连锁分析在常染色体隐性遗传性多囊肾病（ARPKD）家系胚胎植入前遗传学检测（PGT）中的应用价值。方法选取1个ARPKD家系，女方孕期产检发现胎儿有多囊肾行引产，胎儿基因检测为多囊肾/多囊肝病变1基因（PKHD1）复合杂合突变c.10444C > T（父源）和c.4303del（母源），其中c.4303del突变为首次报道。采用多重聚合酶链反应（PCR）和NGS在突变位点两侧2M区域内选择335个信息丰富、紧密连锁的SNP位点作为遗传连锁标记，构建夫妇双方携带基因突变的SNP风险单体型。体外受精后行囊胚培养。对活检获得的滋养层细胞进行全基因组扩增（WGA）后，采用Sanger测序直接检测PKHD1基因突变，采用基于NGS的SNP连锁分析鉴别携带突变的染色体，同时进行拷贝数变异（CNV）分析以进行低深度的染色体非整倍性筛查。结果 6个活检囊胚中有4个为未携带突变的整倍体，有1个携带杂合突变的嵌合体，1个测序数据波动大，无法判断。选择其中1枚未检测到突变的优质整倍体胚胎进行冻融胚胎移植（FET），足月分娩一健康婴儿。结论应用基于NGS的SNP连锁分析进行PGT具有高效稳定的特点，可有效阻断ARPKD在家系中的垂直传递，同时可避免因妊娠非整倍体胚胎而导致的流产问题。该研究也是首次针对PKHD1基因c.4303del突变的PGT报道。

关键词: 常染色体隐性遗传性多囊肾病, 多囊肾/多囊肝病变1基因, SNP连锁分析, 二代测序, 植入前遗传学检测

Abstract:

Objective To investigate the application value of single nucleotide polymorphism （SNP） linkage analysis based on next-generation sequencing （NGS） technology in preimplantation genetic testing （PGT） of families with autosomal recessive polycystic kidney disease （ARPKD）. Methods A family with ARPKD was selected， where the female member had a pregnancy ultrasound revealing polycystic kidney in the fetus. Genetic testing showed compound heterozygous mutations of the polycystic kidney/polycystic liver disease 1 gene （PKHD1）， c.10444C > T （paternal） and c.4303del （maternal）， with the c.4303del mutation being reported for the first time. Targeting the coding region of the PKHD1 gene， 335 high-density tightly linked SNP sites were selected in the upstream and downstream 2M regions using multiplex polymerase chain reaction （PCR） and NGS. The couple's SNP risk haplotypes carrying gene mutations were constructed. After in vitro fertilization， blastocyst culture was performed. Trophoblastic cells obtained from the biopsy were subjected to whole-genome amplification， and NGS was used for linkage analysis and low-depth chromosomal aneuploidy screening of the embryos. Sanger sequencing was used to verify the results of embryo linkage analysis. Results Among the 6 biopsied embryos， 4 were mutation-free and euploid， 1 exhibited heterozygous for the mutation and mosaic while another unstable sequencing data， making it impossible to judge. One of the mutation-free and developmentally healthy euploid embryos was implanted into the maternal uterus， resulting in the full-term delivery of a healthy baby. Conclusion Application of NGS-based SNP linkage analysis in PGT can effectively blocking the vertical transmission of ARPKD within families， while avoiding abortion issues caused by aneuploid embryos. This study is also the first PGT report targeting the PKHD1 gene c.4303del mutation.

Key words: autosomal recessive polycystic kidney disease, polycystic kidney and hepatic disease 1, SNP linkage analysis, next-generation sequencing, preimplantation genetic testing

中图分类号:

R715.5

王凝,郝燕,陈大蔚,章志国,匡丹,张清,尹奕琪,魏兆莲,周平,曹云霞. 胚胎植入前遗传学检测在阻断常染色体隐性多囊肾病家系多囊肾/多囊肝病变1基因新突变遗传的应用[J]. 实用医学杂志, 2024, 40(7): 1006-1010.

Ning WANG,Yan HAO,Dawei CHEN,Zhiguo ZHANG,Dan KUANG,Qing ZHANG,Yiqi YING,Zhaolian WEI,Ping ZHOU,Yunxia CAO. Application research of PGT in blocking the inheritance of novel mutations in the PKHD1 gene in autosomal recessive polycystic kidney disease pedigrees[J]. The Journal of Practical Medicine, 2024, 40(7): 1006-1010.

图/表 1

参考文献 25

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胚胎植入前遗传学检测在阻断常染色体隐性多囊肾病家系多囊肾/多囊肝病变1基因新突变遗传的应用

Application research of PGT in blocking the inheritance of novel mutations in the PKHD1 gene in autosomal recessive polycystic kidney disease pedigrees

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