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

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.

Cell-Free Fetal DNA and Non-Invasive Prenatal Diagnosis of Chromosomopathies and Pediatric Monogenic Diseases: A Critical Appraisal and Medicolegal Remarks

“…We can see that most of them were published in 2021 (13 out of 51 studies), likely due to the relatively young age of the investigation technique. Regarding the reviewed studies, 13 are described in the first section concerning the role of cffDNA in the non-invasive prenatal diagnosis of aneuploidies [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ], 7 are described in the second section (CNV diseases) and the remaining 31 are in the third section concerning diseases with monogenic transmission [ 2 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ].…”

“…Moreover, in these diseases the ultrasonographic diagnosis is often inaccurate and late. In all the cited sources [ 21 , 26 , 34 ] a correct identification of the mutation in affected subjects has been reported. In particular, the study by Yin et al [ 26 ] assessed the fetal genotype of any locus using maternal plasma, through a novel genotyping algorithm named pseudo tetraploid genotyping (PTG).…”

Molecular Characterization of a Rare Case of Monozygotic Dichorionic Diamniotic Twin Pregnancy after Single Blastocyst Transfer in Preimplantation Genetic Testing (PGT)

“…Most noninvasive exclusion of paternal variant reports published to date rely on massively parallel sequencing (MPS) (as recently published for skeletal dysplasia [22]). With an experimental workflow of a few hours, noninvasive prenatal diagnosis by paternal variant exclusion with droplet digital PCR appears to be a valuable option considering MPS costs and turnaround time, as sample processing and sequencing take several days.…”

Tumor Diagnosis of FFPE Sample by Target Genome Sequencing From Multiple Sequencing Platforms

“…Many high-throughput sequencers are compatible with targeted genome sequencing, and have been widely applied in scienti c studies and clinical diagnosis. NextSeq550 system (Illumina), a exible sequencing platform with multiple read length and output con gurations, has been commonly used in WES, transcriptome, as well as targeted sequencing 8,9 . It was reported that the TSO500 panel performed well for variant screening of myeloid neoplasm coupled with the NextSeq550 platform 10 .…”