Non-Invasive Prenatal Diagnosis of Lethal Skeletal Dysplasia by Targeted Capture Sequencing of Maternal Plasma

Abstract

Background: Since the discovery of cell-free foetal DNA in the plasma of pregnant women, many non-invasive prenatal testing assays have been developed. In the area of skeletal dysplasia diagnosis, some PCR-based non-invasive prenatal testing assays have been developed to facilitate the ultrasound diagnosis of skeletal dysplasias that are caused by de novo mutations. However, skeletal dysplasias are a group of heterogeneous genetic diseases, the PCR-based method is hard to detect multiple gene or loci simultaneously, and the diagnosis rate is highly dependent on the accuracy of the ultrasound diagnosis. In this study, we investigated the feasibility of using targeted capture sequencing to detect foetal de novo pathogenic mutations responsible for skeletal dysplasia.

Methodology/principal findings: Three families whose foetuses were affected by skeletal dysplasia and two control families whose foetuses were affected by other single gene diseases were included in this study. Sixteen genes related to some common lethal skeletal dysplasias were selected for analysis, and probes were designed to capture the coding regions of these genes. Targeted capture sequencing was performed on the maternal plasma DNA, the maternal genomic DNA, and the paternal genomic DNA. The de novo pathogenic variants in the plasma DNA data were identified using a bioinformatical process developed for low frequency mutation detection and a strict variant interpretation strategy. The causal variants could be specifically identified in the plasma, and the results were identical to those obtained by sequencing amniotic fluid samples. Furthermore, a mean of 97% foetal specific alleles, which are alleles that are not shared by maternal genomic DNA and amniotic fluid DNA, were identified successfully in plasma samples.

Conclusions/significance: Our study shows that capture sequencing of maternal plasma DNA can be used to non-invasive detection of de novo pathogenic variants. This method has the potential to be used to facilitate the prenatal diagnosis of skeletal dysplasia.

Fig 1. Strategy of variant interpretation.

Fig 2. The distribution of allele frequency and allele depth of real positive foetal specific variants, false positive foetal specific variants and false negative foetal specific variants after the duplicate reads were filtered.

(A) Case 1. (B) Case 2. (C) Case 3. (D) Control case 1. (E) Control case 2. The green circle represents the allele that was shared by the foetal gDNA and plasma DNA (real positive), the black square represents the allele that was detected in foetal gDNA but was not detected in plasma DNA (false negative), and the red triangle represent the allele that was only detected in plasma DNA (false positive).

Fig 3

Computer simulation of foetal specific variants detection in different foetal DNA concentrations and different mean sequencing depth (A) postive predict value. (B) true positve rate.