Limited Clinical Utility of Non-invasive Prenatal Testing for Subchromosomal Abnormalities

Abstract

The use of massively parallel sequencing of maternal cfDNA for non-invasive prenatal testing (NIPT) of aneuploidy is widely available. Recently, the scope of testing has increased to include selected subchromosomal abnormalities, but the number of samples reported has been small. We developed a calling pipeline based on a segmentation algorithm for the detection of these rearrangements in maternal plasma. The same read depth used in our standard pipeline for aneuploidy NIPT detected 15/18 (83%) samples with pathogenic rearrangements > 6 Mb but only 2/10 samples with rearrangements < 6 Mb, unless they were maternally inherited. There were two false-positive calls in 534 samples with no known subchromosomal abnormalities (specificity 99.6%). Using higher read depths, we detected 29/31 fetal subchromosomal abnormalities, including the three samples with maternally inherited microduplications. We conclude that test sensitivity is a function of the fetal fraction, read depth, and size of the fetal CNV and that at least one of the two false negatives is due to a low fetal fraction. The lack of an independent method for determining fetal fraction, especially for female fetuses, leads to uncertainty in test sensitivity, which currently has implications for this technique's future as a clinical diagnostic test. Furthermore, to be effective, NIPT must be able to detect chromosomal rearrangements across the whole genome for a very low false-positive rate. Because standard NIPT can only detect the majority of larger (>6 Mb) chromosomal rearrangements and requires knowledge of fetal fraction, we consider that it is not yet ready for routine clinical implementation.

False negative fetal cell free DNA screening for microdeletion syndromes in the presence of an unbalanced translocation involving monosomy 4p

“…Although this is a single case, our findings are in line with the recent academic validation study by Lo et al , suggesting that rare microdeletions may not be robustly detected using similar genome‐wide counting‐based cfDNA assays, particularly in samples with relatively low fetal cfDNA fraction. Wolf‐Hirschhorn syndrome is associated with terminal deletions of 4p as small as 2 Mb .…”

“…In the current pregnancy, this assay was reported as normal despite the presence of the same unbalanced translocation in the fetus. To our knowledge, while false negative results have been found in the academic setting, and are to be expected in any screening assay, such false negative results from commercially available microdeletion testing have not yet been reported in the literature. Awareness of the limitations of these assays is important both for provider education and for offering laboratories.…”

Toward an Ethically Sensitive Implementation of Noninvasive Prenatal Screening in the Global Context

“…The inclusion of increasingly rare conditions in cfDNA panels will inevitably increase the false positive rate of cfDNA screening, potentially limiting the primary benefit of cfDNA screening in reducing the need for diagnostic testing. 37, 38 This may be especially problematic in low resource areas with limited access to the skilled diagnostic practitioners required to confirm cfDNA results. Significant differences exist between these newly added conditions and the currently-screened trisomies, leading some to argue that highly disparate clinical realities are now being inappropriately lumped together and raising additional challenges to patient counseling.…”

Implementation of non‐invasive prenatal testing by semiconductor sequencing in a genetic laboratory

“…Recent studies have shown that with NIPT, also other autosomal aneuploidies (other than trisomy 13, 18 and 21) and even segmental chromosomal aberrations can be detected . Further validation and clinical studies in larger cohorts are however warranted to assess the added value, sensitivity and specificity of these results …”