Comprehensive Cytogenetic Analysis Reveals Mosaicism in Newborn with Negative Prenatal Down Syndrome Screening: A Case Report

Introduction

Down syndrome (DS), or trisomy of autosome 21, is one of the most common chromosomal disorders associated with intellectual disability. Prenatal screening is a proactive approach to identify potential fetuses with common chromosomal abnormalities, primarily DS. Over recent years, prenatal screening for chromosomal disorders, particularly DS, has undergone extraordinary development. These screenings now encompass a wide array of options, including biochemical and ultrasound markers during the first and second trimester, third trimester ultrasound examination when needed, and the increasingly prevalent non-invasive prenatal testing (NIPT) [1,2].

Limitations of all types of prenatal screening for chromosomal abnormalities include false-positive and false-negative results. False-positive cases are typically identified through subsequent invasive prenatal diagnostic procedures, while false-negative cases can go undetected, particularly in cases lacking abnormal ultrasound findings, or when pregnant women decline further testing in the later weeks of pregnancy, due to a misunderstanding of the limitations of previous screenings.

Both the false-positive and false-negative results across all screening modalities can be attributed to various factors, including technical issues, computational errors, biological variability, and staff errors. One of the key biological factors contributing to diagnostic limitations is chromosomal mosaicism. Mosaicism refers to a condition in which 2 or more cell clones with different genetic compositions originate from a single zygote within the same individual [3]. Mosaic variants of DS account for approximately 1% to 4% of all cases and involve the presence of 2 or more cell clones in a carrier, with 1 having trisomy 21 [3]. Unlike full trisomy DS, which is more common in males, mosaic DS tends to exhibit a higher prevalence in females across different populations. This pattern suggests that in utero selection against the male embryo can play a role in the development of mosaic fetuses [4].

A significant challenge in postnatal analysis of newborns with suspected DS missed during prenatal screening lies in the incomplete cytogenetic or cytogenomic profiling of the patients. Relying exclusively on the analysis of phytohemagglutinin (PHA)-stimulated blood cells limits the ability to comprehensively characterize a patient’s karyotype, because it analyzes predominantly T lymphocytes [5]. The International Mosaic Down Syndrome Association recommends complementing conventional T lymphocyte karyotyping by fluorescence in situ hybridization (FISH) or chromosomal microarray in patients who present with clinical features of the condition but have a normal karyotype [6]. Nonetheless, the detection of the full-form aneuploidy or a normal karyotype by karyotyping on PHA-stimulated lymphocytes does not rule out the possibility of mosaicism in other tissues. Such tissue-specific patterns have long been well known for gonosomal disorders [for example, 7] and have also been detected in cases of trisomy 21 [8–10].

We present a case study of a newborn with DS that was missed during prenatal screening. The screening process included maternal serum analyte tests and ultrasound examinations during the first and second trimesters of pregnancy. We performed a combined cytogenetic approach using classical GTG-banding techniques on stimulated T lymphocyte blood samples and FISH on unstimulated blood nuclei and buccal swabs. This in-depth cytogenetic analysis allowed us to compare chromosomal alterations in tissues derived from both the different and the same embryonic layers, providing insight into the potential tissue-specific and intercellular mosaicism for chromosome 21, which could be the biological reason for false-negative screening results. Based on our findings, we propose a more comprehensive cytogenetic examination for newborns with negative screening results but clinical features suggestive of DS, to ensure a complete genetic assessment.

Case Report

A 9-day-old female newborn underwent karyotyping at the EcoSense laboratory due to suspected DS, based on the clinical features observed at birth. The laboratory did not have access to the complete clinical data on the proband. The mother was 39 years old during the pregnancy and was experiencing infertility prior to conception, with no history of miscarriages. The newborn was the mother’s first child. During the pregnancy, the mother underwent first- and second-trimester biochemical and ultrasound screenings. The β-hCG and PAPP-A levels were 1.022 MoM and 0.947 MoM, respectively, with fetal nuchal translucency measured to be 2 mm at 13.0 weeks of gestation. The estimated personal risk for DS was 1: 1866. No ultrasound markers of chromosomal abnormalities were detected in the first and second trimesters, and the third-trimester ultrasound was not performed. The mother declined to undergo NIPT.

Postnatal karyotyping of 77 cells from 2 independent PHA-stimulated T lymphocyte cultures confirmed trisomy 21 in all analyzed metaphase plates (Figure 1). The karyotype formula according to the International System for Human Cytogenomic Nomenclature (ISCN) 2024 [11] was 47,XX,+21.

To investigate potential tissue-specific and intercellular mosaicism for chromosome 21, FISH analysis was performed on the available tissues. For this purpose, unstimulated lymphocyte nuclei were used to assess both T- and B-lymphocyte populations, derived from the mesodermal embryonic layer, as well as buccal epithelium, derived from the endodermal embryonic layer. The form (split, diffuse, compact) and the distribution of fluorescent signals specific to each probe were analyzed according to the manufacture’s counting guidelines (Abbott Molecular, USA). Overlapping nuclei were excluded from signal enumeration.

FISH analysis of unstimulated lymphocyte nuclei and buccal smears revealed mosaicism, with approximately 20% of cells in both tissues displaying disomy for chromosome 21 (Figure 2). The karyotype formula according to ISCN 2024 [11] for unstimulated lymphocyte is

and for buccal swab is

Maternal and paternal blood samples were not available, precluding determination of the parental origin and the type of error, meiotic or mitotic, responsible for the extra autosome.

Discussion

Karyotyping of PHA-stimulated lymphocytes is the first-tier method used to assess the cytogenetic status of newborns with suspected chromosomal abnormalities missed during prenatal screening. In routine practice, 20 metaphases are typically analyzed per patient, which allows sufficient resolution to evaluate the entire chromosomal set in the absence of crossover events, either between chromatids or between chromosomes [12]. To exclude mosaicism, it is recommended to analyze a minimum of 30 metaphases, which allows detection of low-level mosaicism (10%) [12,13]. In cases in which the minor clone constitutes less than 5% (low-grade or cryptic mosaicism), analysis of a minimum of 50 metaphases is needed [6,13,14].

We showed that conventional cytogenetic examination of T lymphocytes can provide an incomplete representation of the chromosomal status in newborns with suspected chromosomal abnormalities. In our study, 77 metaphase plates from 2 independent cell cultures were analyzed, enabling the exclusion of low-level and cryptic mosaicism with 99% and 95% confidence, respectively [14]. However, FISH identified a disomic clone for chromosome 21 in approximately 24% of nuclei from uncultured lymphocytes. It is well established that cell clones with specific karyotypes can gain a proliferative advantage in culture [15,16]. For instance, in cases of partial tetrasomy 12p associated with Pallister-Killian syndrome, cultured blood samples often display a normal karyotype. By contrast, uncultured blood cells or slower-dividing cultures, such as fibroblasts, can reveal the presence of tetrasomy 12p clones [15]. In the present study, a similar phenomenon might have occurred, in which trisomy 21 clones gained a selective advantage during the culture of newborn blood cells. Furthermore, the analysis of unstimulated lymphocytes encompasses T lymphocytes and B lymphocytes, with the B lymphocytes being less responsive to mitogenic stimulation with PHA [5,17]. The discordance in intercellular karyotype results shown in our case shows that mosaicism could be more complex than what is revealed by conventional karyotyping. This complexity could be related to the biological particularities of the analyzed cells and the limitations imposed by the widely used mitogen PHA, rather than to technical limitation of the FISH method.

Mosaicism is known to be a dynamic process during ontogeny, with the proportions of cells exhibiting normal and abnormal karyotypes changing over time. For patients with DS, the percentage of abnormal clones in cultured and uncultured lymphocytes decreases with age, while the mosaicism level in buccal smears and fibroblast cultures remains stable [18,19]. This underscores the importance of cytogenetic analysis of buccal epithelial cells as an additional tissue source for investigating mosaic variants of autosomal trisomies. Moreover, buccal smear collection is a non-invasive procedure, which is especially important for diagnostic purposes in newborns.

A disomic clone for chromosome 21 was also identified in buccal smears of our proband, with the proportion of normal and abnormal cells in uncultured blood and buccal samples being approximately equivalent. These findings suggest that the loss of one chromosome 21 occurred prior to the differentiation of embryonic layers.

It is well established that in fetuses with DS, the formation of the syncytiotrophoblast from the cytotrophoblast is impaired, resulting in abnormal chorionic gonadotropin signaling. This disruption results in elevated levels of β-hCG in maternal blood, which can be detected during prenatal biochemical screening [20]. In our case, first-trimester screening revealed normal ranges of β-hCG and PAPP-A protein levels. Nonetheless, given the advanced reproductive age of the proband’s mother, a meiotic origin for the chromosomal abnormality cannot be ruled out. It is possible that a trisomic clone was present in the chorionic villus cells in a mosaic state, which could represent an additional biological factor contributing to the false-negative result in this case. However, the chromosomal status of extraembryonic tissue could not be determined, as the mother declined NIPT during pregnancy, and placental tissue was not collected for genetic analysis after delivery.

Analysis of intercellular and tissue-specific mosaicism is especially important in studies of genotype-phenotype correlations. This information can also be essential for genetic counseling and prognosis. In general, patients with mosaic DS who have a lower proportion of trisomic cells tend to exhibit milder phenotypic features, fewer congenital abnormalities, and better cognitive development than do those with higher levels mosaicism or full trisomy 21 [3,21]. Early studies indicated that the presence of a 50% trisomic cell line could result in the same intellectual problems as in patients with full trisomy 21 [22]. Currently, it is known that patients with the mosaic variant of DS display varying phenotypic manifestations, depending on the percentage of trisomic cells and the origin of the mosaic cell line [21]. For instance, it was shown that a high proportion of trisomic cells in the buccal mucosa (53% or more) is associated with a lower IQ, whereas the presence of heart defects significantly correlates with higher levels of mosaicism in lymphocytes (54% or more cells with trisomy) [21]. In rare cases, patients with low-level mosaicism of trisomy 21 (10% abnormal cells) in PHA-stimulated T lymphocytes, but without clinical manifestations of DS, have shown early onset of Alzheimer disease, typical for full trisomy 21 [23]. However, in the latter case, it is possible that the patient can also have mosaicism in other tissues that were not included in the study. These tissues can have a different proportion of trisomic cells, as demonstrated in a case study in which both the blood and buccal swab samples were analyzed [24].

In our case, the laboratory did not have a clearly defined description of the proband’s phenotype, which limited the ability to performed phenotype-genotype correlations. According to prenatal ultrasound data, no markers of aneuploidy were detected during the first and second trimesters. However, certain developmental anomalies can become more apparent during later stages of embryonic development. Third trimester ultrasound screening was not conducted in our case.

This case highlights the challenges in diagnosing mosaic DS and further emphasizes the limitations of current prenatal screening methods. Comprehensive postnatal cytogenetic investigation of newborns with trisomy 21 missed during prenatal screening is crucial for identifying the biological causes of false-negative results. Such findings can help overcome the limitations of the current technological platforms and improve existing algorithms. Thus, according to the American College of Medical Genetics and Genomics, NIPT is strongly recommended over traditional screening programs for all pregnant women [2]. The ability of NIPT to detect mosaicism for chromosome 21 varies depending on the technology platform and distribution of abnormal cell clones and can be false-negative in rare cytogenetic mosaic variant of DS [25–27]. According to some published studies, the level of detectable mosaicism ranges from 15% to more than 30% [26,27]. Despite its limitations, NIPT is a highly accurate prenatal screening method, due to its analysis of cell-free fetal DNA fragments in maternal blood. It is especially recommended for pregnant women of advanced reproductive age, like in our case. In some countries, NIPT has become available as the primary screening option for chromosomal aneuploidies in all pregnant women or high-risk groups and is covered by public health programs or publicly funded financed systems [28]. However, in countries where NIPT is currently offered only as a self-financed commercial prenatal screening for fetal aneuploidies, developing a national program to make non-invasive genetic screening more affordable and accessible should be a priority.

Therefore, it is essential to develop new, additional non-invasive diagnostic methods. For instance, recent studies have identified genetic biomarkers associated with congenital heart abnormalities [29,30], which are among the most common ultrasound markers for chromosomal abnormalities, including DS. Although prenatal ultrasound is a common practice for identifying congenital abnormalities during pregnancy, the detection rate of cardiac abnormalities varies across different centers and is influenced by several factors, including the equipment quality, type of anomaly, technician’s expertise, and specific clinical guidelines followed [29]. Enhancing diagnostic capabilities for these biomarkers could significantly improve prenatal screening outcomes, particularly for high-risk groups, such as women of advanced reproductive age.

Conclusions

Our results demonstrate that the combined approach of karyotyping with FISH on unstimulated lymphocytes and buccal smear nuclei enhances diagnostic accuracy in newborns with suspected chromosomal abnormalities missed during traditional prenatal screening. This integrated approach might be useful for research concerning genotype-phenotype correlations and evaluation of age-acquired mosaicism and its association with age-related comorbidities in individuals with trisomy 21. Further studies are needed to determine whether there is a relationship between mosaicism – its level, tissue specificity, and intercellular distribution – and false-negative results in prenatal screening based on biochemical and ultrasound examinations.