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Wang CY, Tang YA, Lee IW, et al. Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia. BMC Med Genomics. 2021;14:212doi: 10.1186/s12920-021-01063-1.
Wang, C. Y., Tang, Y. A., Lee, I. W., Chang, F. M., Chien, C. W., Pan, H. A., & Sun, H. S. (2021). Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia. BMC medical genomics, 14212. https://doi.org/10.1186/s12920-021-01063-1
Wang, Ching-Yuan, et al. "Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia." BMC medical genomics vol. 14 (2021): 212. doi: https://doi.org/10.1186/s12920-021-01063-1
Wang CY, Tang YA, Lee IW, Chang FM, Chien CW, Pan HA, Sun HS. Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia. BMC Med Genomics. 2021 Nov 17;14:212. doi: 10.1186/s12920-021-01063-1. PMID: 34789231; PMCID: PMC8600686.
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BMC Med Genomics. 2021 Nov 17;14:212. doi: 10.1186/s12920-021-01063-1.

Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia.

BMC medical genomics

Ching-Yuan Wang, Yen-An Tang, I-Wen Lee, Fong-Ming Chang, Chun-Wei Chien, Hsien-An Pan, H Sunny Sun

Affiliations

  1. Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, 1 University Road, Tainan, 70101, Taiwan.
  2. Center for Genomic Medicine, Innovation Headquarters, National Cheng Kung University, Tainan, Taiwan.
  3. FMC Fetal Medicine Center, Tainan, Taiwan.
  4. AN-AN Women and Children Clinic, Tainan, Taiwan.
  5. Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, 1 University Road, Tainan, 70101, Taiwan. [email protected].
  6. Center for Genomic Medicine, Innovation Headquarters, National Cheng Kung University, Tainan, Taiwan. [email protected].

PMID: 34789231 PMCID: PMC8600686 DOI: 10.1186/s12920-021-01063-1

Abstract

BACKGROUND: Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy.

METHODS: An ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated.

RESULTS: The established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel.

CONCLUSIONS: An expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice.

© 2021. The Author(s).

Keywords: Amplicon-based targeted sequencing; Noninvasive prenatal testing (NIPT); Precision medicine; Skeletal dysplasia

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