Placental studies elucidate discrepancies between NIPT showing a structural chromosome aberration and a differently abnormal fetal karyotype

Abstract Objective Placental cytogenetic studies may reveal the origin of discordant noninvasive prenatal testing (NIPT). We performed placental studies to elucidate discordances between NIPT showing a structural chromosome aberration and the fetus having a different chromosome aberration in three cases. Method Diagnostic testing with genomic SNP microarray was performed in three cases with NIPT showing a duplication on 4q (case 1), a terminal deletion of 13q (case 2), and a terminal deletion of 15q (case 3). Placental studies involved SNP array analysis of cytotrophoblast and mesenchymal core of chorionic villi of four placental quadrants. Clinical follow‐up was performed as well. Results Amniotic fluid revealed a different structural chromosome aberration than predicted by NIPT: a terminal 2q deletion (case 1), a segmental uniparental isodisomy of 13q (case 2), and a terminal duplication of 15q and of 13q (case 3). Placental studies revealed the aberration detected with NIPT in the cytotrophoblast, whereas the fetal karyotype was confirmed in the placental mesenchymal core. Conclusion Our study shows that targeted cytogenetic investigations for confirmation of NIPT showing a microscopically visible structural chromosome aberration should be avoided, since another aberration, even a submicroscopic one or one involving another chromosome, may be present in the fetus.

large, microscopically visible fetal chromosome aberrations of greater than 10 to 15 Mb (eg, subchromosomal aberrations). The detection of specific submicroscopic chromosome aberrations (eg, microdeletions and microduplications, typically less than 5 Mb in size) also have been described, and nowadays, these are sometimes included in commercial NIPT-kits.
Since the fetal part of the cell-free DNA is derived from the cytotrophoblast (CTB) of chorionic villi (CV), NIPT in fact detects placental chromosome aberrations. Although the chromosome constitution of placenta and fetus are expected to be the same in most cases, it is known, mainly from CV studies, that discrepancies may occur in 1% to 2% of CVS, at least in a high-risk population. 4 Confined placental mosaicism (CPM) is the main reason for discordant NIPT results. 5 In a previous study, we showed that about 15% of rare autosomal trisomies (RAT, autosomal trisomies different from trisomies of chromosomes 13, 18, and 21) that were detected with NIPT in pregnancies with abnormal first trimester combined (FTC) test results were confirmed in the fetus, but the rest mainly showed to be confined to the placenta. 1 In contrast, in that same study, it was shown that 50% (six out of 12) of large, microscopically visible (greater than 10 Mb), structural chromosome aberrations that were detected with NIPT, were confirmed in the fetus. This is in line with other papers showing a confirmation rate of 50% to 62%. 2,6 Follow-up investigations after abnormal NIPT often are limited to fetal, and depending on the NIPT result, also maternal cytogenetic investigations, and if this reveals normal results, the NIPT is called In this paper, we present the results of placental follow-up investigations that were performed in three cases of abnormal NIPT showing a structural chromosome aberration and another abnormal fetal karyotype involving a different structural aberration in an effort to elucidate the observed discrepancies.

| MATERIALS AND METHODS
We present three cases in which NIPT revealed a structural chromosome aberration and in which fetal, placental, and maternal cytogenetic follow-up investigations during and after pregnancy were performed in order to elucidate the discrepancies that were found between the abnormal NIPT (partial duplication of 4q, a partial deletion of 13q, and a partial deletion of 15q), and differently abnormal fetal karyotype (with respectively a terminal deletion of 2q, a segmental uniparental disomy of 13q and a partial duplication of 13q and 15q).
In all cases, NIPT was performed as part of the Dutch Trident 1 study (Trident = Trial by Dutch laboratories for Evaluation of NIPT

| Fetal and parental cytogenetic follow-up studies
The results of cytogenetic investigations of AF, cord blood, and/or buccal swab after birth and of parental blood for confirmation of an abnormal NIPT result are shown in Table 1. In all three cases, another chromosome aberration than the one predicted by NIPT was found in the fetus: • In case 1, NIPT detected a duplication on 4q ( Figure 1A), while the fetus had a terminal deletion of 2q.
• In case 2 a deletion on 13q as detected with NIPT ( Figure 1B) showed to be a segmental uniparental isodisomy (UPiD) of maternal origin of the terminal part of 13q in fetal cells. The PCCA gene in this region was screened for mutations; none were found.
• In case 3 with a deletion of distal 15q in NIPT ( Figure 1C), a mosaic duplication of the distal part of the long arms of chromosomes 13 and 15 was found in AF cells.

| Placental cytogenetic confirmatory testing
In an effort to elucidate the discordances between NIPT and prenatal diagnosis, placental studies were performed. In all three cases, analysis of placental CTB revealed the chromosome aberration that was found with NIPT, while the chromosome constitution of the MC was representative for that of the fetus:

| Clinical outcome
Clinical outcome data are shown in  The other cell line is the one with approximately 25-Mb ROH on 13q31.3q34 that was also detected in MC, AF, cord blood, and buccal mucosa.   in one CTB biopsy that was not seen prenatally (with NIPT or invasive testing). This phenomenon of extra chromosome anomalies in the placenta was recently described in 2/10 placentas that were investigated in order to confirm abnormal NIPT involving a numerical chromosome aberration. 9 Unfortunately, it could not be investigated whether there is a cytogenetic association between the duplication of chromosome 4q that was detected with NIPT and the terminal deletion of 2q (case 1).
The 4q duplication was only present in the CTB of two biopsies, and these samples were not cultured but used entirely for DNA isolation for SNP array (therefore, no chromosome preparations were available).
However, it is possible that the 4q was "captured" by the 2q terminal deletion for telomere stabilization, at least in one of the early embryonic cells that was allocated to the CTB. It was recently shown that distinct stabilizing events, telomere healing (eg, de novo telomere addition mediated by telomerase) and telomere capture from a different chromosome, resulting in a derivative chromosome, of the same terminal deletion can occur in different early embryonic cells. 15 Mb was present in the majority of cells. Only low-level mosaicism of a larger 56-Mb deletion was present in one of the biopsies, which probably did not result in a sufficient contribution to the cfDNA pool in maternal plasma, so that it remained undetected with our NIPT approach, characterized by a resolution of 10 to 15 Mb. 17 In the era of NIPT, which investigates cfDNA that originates from the CTB of CV, placental cytogenetic investigations are in the spotlight again. Placental studies used to be frequently performed after the introduction of CVS in the 80s of last century, 18 and that led to an exponential increase of our knowledge of CPM and its clinical relevance. In our opinion, placental studies for confirmation of an abnormal NIPT result are important for several reasons: • If a placental origin is proven, another source for the abnormal cfDNA, such as a maternal tumor, can be excluded.
• For reassurance of the parents that may be anxious after an abnormal NIPT despite normal cytogenetic results from AF.
• For increased knowledge of the origin of abnormal NIPT and its associated clinical impact.
• For better interpretation of abnormal results and therefore improved pre-as well as posttest counseling.
• Finally, for better insight into the true performance of the NIPT test, probably reaching a positive predictive value of 100% if all possible sources of the cfDNA would be investigated.
The main conclusion of this study is that the use of targeted cytogenetic investigations for confirmatory diagnostic testing of NIPT showing a structural chromosome aberration should be avoided since another chromosome aberration, even involving another chromosome, may be present in the fetus. Moreover, also the use of karyotyping should be discouraged and replaced by preferably SNP array since a submicroscopic structural aberration or segmental UPD may be present in the fetus even though the NIPT predicts a microscopically visible chromosome aberration.