Fetal mosaicism as a cause of false-negative noninvasive prenatal DNA screening for chromosome 21 aneuploidies

Introduction

Noninvasive prenatal DNA screening (NIPS) has been available for fetal aneuploidy detection since 2011. Large-scale studies declare high sensitivity and specificity of NIPS for common aneuploidies detection especially for trisomy 21 [1]. Nevertheless, the test cannot reach diagnostic accuracy due to a possibility of false-positive and false-negative results [2]. Discordant NIPS results occur due to biological reasons such as confined placental mosaicism, maternal copy number variations, vanished twins, maternal cancer, true fetal mosaicism. False-negative results are mainly explained by a true fetal mosaicism [2].

Two mechanisms underlying mosaicism formation are described. They both involve non-disjunction errors during meiosis followed by trisomy rescue or during mitotic cell division. Regarding the first mechanism, non-disjunction usually occurs in maternal meiosis and results in an abnormal zygote. The normal cell line appears in a subsequent mitotic division with the loss of one of the extra chromosomes. Both normal and trisomic cell lines are detected with cytogenetic analysis. Regarding the second mechanism, non-disjunction happens in an initially normal zygote and results in three cell lines: the trisomic, the normal and the monosomic cell lines. It is believed that autosomal monosomic cell line rapidly disappears and only two cell lines are detected with cytogenetic analysis.

The proportion of fetal DNA in maternal plasma is a crucial parameter for test performance [3]. Low fetal DNA fraction leads to very small and undetectable overrepresentation of trisomic chromosome. The majority of NIPS providers measure and report fetal DNA fraction as a test quality control parameter. Mosaicism in fetal cells leads to a reduction of trisomic DNA fraction while overall measured fetal DNA fraction can stay above the required limit resulting in false-negative calls. False-negative NIPS calls lead to a delayed diagnosis of fetal aneuploidy or an unexpected delivery of a baby with an aneuploidy [4-6]. A recent review of discordant NIPS results has shown that a significant part of false-negative cases remains unexplained [2].

This study describes an unusual case of falsenegative trisomy 21 NIPS result due to true fetal mosaicism with three different cell lines. Patient provided approval to use the obtained results for research purposes and scientific publications.

Case Presentation

The 35-year-old woman (gravida 3, para 1) referred to the laboratory for genetic counseling and NIPS. The results of first-trimester screening indicated that the pregnancy was at high risk of trisomy 21. The patient was proposed invasive procedure and informed about restrictions of NIPS. Despite that, she refused invasive diagnostics and preferred to make noninvasive test. NIPS was performed at 15 weeks gestation and showed low risk for all chromosomes. Fetal fraction estimated from Y chromosome coverage was 10%.

After the birth baby was suspected to have Down syndrome. Blood was collected in EDTA tube and delivered to the laboratory. Molecular karyotyping was performed using CytoScan Optima Array (Affymetrix, Santa Clara, CA) and showed full trisomy 21 result.

To investigate the presence of mosaicism, an interphase FISH analysis of 101 white blood cells was performed with AneuVision (Abbott Molecular) probes for 5 chromosomes (13, 18, 21, X, Y). It revealed 3 signals for chromosome 21 in 77 nuclei, 2 signals in 18 nuclei and 1 signal in 6 nuclei. They corresponded to mosaic karyotype with trisomy 21 (76%), monosomy 21 (6%) and disomy 21 (18%).

Reanalysis of sample aliquot confirmed previous NIPS result. It showed low risk for all chromosomes and 10% fetal fraction. Sample identity was ascertained by means of SNP markers.

The blood was collected one more time at the age of 2 months in heparin tube for karyotyping, FISH analysis was also repeated. Conventional karyotyping of 100 cells revealed mosaic karyotype mos 47,XY + 21[94]/46,XY[6] with no monosomic clones. FISH analysis of 300 peripheral blood cells confirmed the presence of 3 cell lines with 3 signals for chromosome 21 in 256 cells (85%), two signals in 56 cells (13%) and 1 signal in 5 cells (2%).

Discussion

Although it is believed that in case of a mitotic non-disjunction autosomal, monosomic cell line rapidly disappears, we have demonstrated that some cells with monosomy 21 can survive until delivery. Unfortunately, placental tissues were not available for analysis, so we were not able to assess cell line distribution in the placenta. We suppose that the presence of DNA from monosomic clones along with DNA from cells with trisomy 21 could mask trisomic signal and lead to false-negative call.

Our study shows that mosaicism detection is significantly affected by confirmation method. Despite the presence of SNP probes in the arrays, molecular karyotyping did not show any features of mosaicism. FISH analysis of the same blood sample showed 3 different cell lines.

Conventional karyotyping of a peripheral blood sample collected at the age of 2 months did not detect any monosomic clones. Moreover, only 6% of analyzed cells had a normal karyotype, such cells could be missed with the examination of 10-20 cells as recommended for routine karyotyping by the European Cytogeneticists Association. The absence of monosomic clones in conventional karyotyping results could be due to cell culturing bias. FISH analysis of the peripheral blood sample collected at the age of 2 months revealed a shift in cell lines ratio. The shift in cell lines ratio may be caused by lower monosomic clones’ survival rate. The proportion of these cell lines can vary in different tissues and change during gestation. The presence of monosomic clones elimination during gestation and the impact of karyotyping method on obtained results can clarify unexplained false-negative cases [2].

This case illustrates that negative result does not rule out the possibility of a fetus affected with trisomy and shows the significance of the first-trimester screening results and restrictions of NIPS in case of high-risk pregnancies.

The study was performed with the support of the Ministry of education and science of the Russian Federation. (Agreement № 14.607.21.0136, project ID RFMEFI60715X0136)