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Sci Rep. 2016 May 6;6:25488. doi: 10.1038/srep25488.

Identification of PKD2 mutations in human preimplantation embryos in vitro using a combination of targeted next-generation sequencing and targeted haplotyping.

Chen SC1,2, Xu XL3,4, Zhang JY1,2, Ding GL1,2, Jin L1,2, Liu B5, Sun DM3,4, Mei CL6, Yang XN3,4, Huang HF1,2,5, Xu CM1,2,5.

Author information

1
Institute of Embryo-Fetal Original Adult Disease Affiliated to Shanghai Jiao Tong University School of Medicine, 910 Hengshan Road, Shanghai 200030, P.R. China.
2
International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, 910 Hengshan Road, Shanghai 200030, P.R. China.
3
BGI-Shenzhen, Shenzhen, 518083, P.R. China.
4
Hangzhou Genomics Institute, 478 Wensan Road, Hangzhou 310012, P.R. China.
5
Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of Education, 1 Xueshi Road, Hangzhou 310006, P.R. China.
6
Division of Nephrology, Kidney Institute of CPLA, Changzheng Hospital, Second Military Medical University, Shanghai,200003, P.R.China.

Abstract

Here, we evaluate the applicability of a new method that combines targeted next-generation sequencing (NGS) with targeted haplotyping in identifying PKD2 gene mutations in human preimplantation embryos in vitro. To achieve this goal, a proband family with a heterozygous deletion of c.595_595 + 14delGGTAAGAGCGCGCGA in exon 1 of the PKD2 gene was studied. A total of 10 samples were analyzed, including 7 embryos. An array-based gene chip was designed to capture all of the exons of 21 disease-related genes, including PKD2. We performed Sanger sequencing combined with targeted haplotyping to evaluate the feasibility of this new method. A total of 7.09 G of data were obtained from 10 samples by NGS. In addition, 24,142 informative single-nucleotide polymorphisms (SNPs) were identified. Haplotyping analysis of several informative SNPs of PKD2 that we selected revealed that embryos 3, 5, and 6 did not inherit the mutation haplotypes of the PKD2 gene, a finding that was 100% accurate and was consistent with Sanger sequencing. Our results demonstrate that targeted NGS combined with targeted haplotyping can be used to identify PKD2 gene mutations in human preimplantation embryos in vitro with high sensitivity, fidelity, throughput and speed.

PMID:
27150309
PMCID:
PMC4858692
DOI:
10.1038/srep25488
[Indexed for MEDLINE]
Free PMC Article

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