2024-01-22 03:03:58
He fetal genetic diagnosis is fundamental in the prenatal careand recent advances in prenatal trio-exome sequencing (i.e., simultaneous sequencing of the coding regions of the genome of the fetus, mother and father) propose diagnostic benefits. However, due to the invasive nature of fetal sampling, its use is limited to fetuses with identifiable structural abnormalities. This limitation leaves many monogenic disorders undiagnosed because they do not manifest in the prenatal period, resulting in a large number of newborns with fatal pediatric disorders and seriously debilitating.
The development of non-invasive fetal testing using circulating DNA of a maternal blood sample has revolutionized prenatal screening, but its application has been limited mainly to chromosomal alterations due to the low resolution of existing tests.
To address these limitations, they developed a non-invasive prenatal screening using trio-exome deep sequencing. This comprehensive method has a high sensitivity to detect fetal single nucleotide variants, small insertions and deletions, high copy number mutations and chromosomal aneuploidies from circulating DNA obtained from a maternal blood sample (Figure 1). This proof-of-concept approach leverages error-corrected ultra-deep trio-exome sequencing to detect fetal variants once more (new, non-inherited mutations) with high precision. These are the main cause of severe pediatric developmental disorders.
Figure 1: Performance and workflow of non-invasive prenatal screening with trio-exome deep sequencing.
In this study, they recruited 36 pregnant women and their partners. The key inclusion criteria were a measurement of the nuchal translucency of at least 5 mm, at least one fetal anomaly identified during the first or second trimester of gestation, or both. They made a ultra-deep exome sequencing in free DNA extracted from maternal plasma, achieving high sequencing coverage per sample. A custom process was used to accurately name and filter variants, incorporating error correction using unique molecular identifiers, site-specific noise modeling, and fetal fraction estimation that ranged from 3.72-19%.
Trio-exome analysis, including maternal plasma and matched parental blood samples, was performed for the identification of novel fetal and paternally inherited variants. The performance of non-invasive prenatal screening using trio-exome deep sequencing was evaluated using the results obtained with this new method, in comparison with invasive whole-exome sequencing, whole-genome sequencing, and analysis of microarrays chromosomes from the chorionic villi or amniotic fluid samples, for all cases.
The following were identified fetal pathogenic variants once morewhich are considered clinically relevant in pregnancy: four single nucleotide mutations, one small deletion, two autosomal trisomies, one sex chromosome aneuploidy, two high copy number variants, and one unbalanced translocation. All mutations detected with this non-invasive method were confirmed in fetal samples obtained invasively. These aligned with the fetal phenotype and provided a genetic diagnosis in 11 of the 36 pregnancies. No additional variants were identified by invasive analyzes in the 36 cases. Therefore, complete agreement is observed between non-invasive and invasive prenatal analysis, with 100% detection of pathogenic variants. once more.
To assess representation of the fetal exome in circulating DNA, they expanded the analysis to encompass all paternally inherited fetal sequence variants within the coding region. The average sensitivity of the new method for detecting these mutations in all protein-coding genes was 95.12%.
In a subset of plasma samples, this new technique revealed a small number of variants of unknown origin, which were not identified in the corresponding invasively obtained samples. However, none of these were considered clinically relevant or required further testing.
In this non-invasive method, samples from the parents were included, which allows carrier screening in both parents to identify pregnancies that are at high risk for recessive disorders. This testing approach demonstrates that it can accurately determine fetal inheritance of paternal mutations in the event that the mother is not a carrier. Confirming the presence of a maternally inherited variant in the fetus would require invasive testing.
This type of prenatal screening provides a means to detect, non-invasively and comprehensively, a wide range of genetic disorders without posing a risk to the fetus or the mother. Integrating this technique into routine prenatal care along with ultrasound screening would provide an opportunity to improve early detection rates, reduce the number of invasive procedures, and facilitate rapid interventions. Although large studies are needed to test this hypothesis, the findings of this investigation (Figure 1B) would predict a low false positive rate. All positive results must be confirmed by prenatal invasive diagnostic testing. With a turnaround time of approximately 7 days (Figure 1C), non-invasive prenatal screening by trio-exome deep sequencing provides timely results.
The findings support further investigation of deep exome sequencing of circulating DNA to detect pathogenic variants in fetuses with and without identifiable structural abnormalities.
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