Prenatal diagnosis of BACs‐on‐Beads assay in 1520 cases from Fujian Province, China

Abstract Background The aim of this study was to evaluate the application of BACs‐on‐Beads (BoBs™) assay for rapid detection of chromosomal abnormalities for prenatal diagnosis (PND). Methods A total of 1520 samples, including seven chorionic villi biopsy samples, 1328 amniotic fluid samples, and 185 umbilical cord samples from pregnant women were collected to detect the chromosomal abnormalities using BoBs™ assay and karyotyping. Furthermore, abnormal specimens were verified by chromosome microarray analysis (CMA) and fluorescence in situ hybridization (FISH). Results The results demonstrated that the success rate of karyotyping and BoBs™ assay in PND was 98.09% and 100%, respectively. BoBs™ assay was concordant with karyotyping for Trisomy 21, Trisomy 18, and Trisomy 13, sex chromosomal aneuploidy, Wolf–Hirschhorn syndrome, and mosaicism. BoBs™ assay also detected Smith–Magenis syndrome, Williams–Beuren syndrome, DiGeorge syndrome, Miller–Dieker syndrome, Prader–Willi syndrome, Xp22.31 microdeletions, 22q11.2, and 17p11.2 microduplications. However, karyotyping failed to show these chromosomal abnormalities. A case of 8q21.2q23.3 duplication which was found by karyotyping was not detected by BoBs™ assay. Furthermore, all these chromosomal abnormalities were consistent with CMA and FISH verifications. According to the reports, we estimated that the detection rates of karyotyping, BoBs™, and CMA in the present study were 4.28%, 4.93%, and 5%, respectively, which is consistent with the results of a previous study. The respective costs for the three methods were about $135–145, $270–290, and $540–580. Conclusion BoBs™ assay is considered a reliable, rapid test for use in PND. A variety of comprehensive technological applications can complement each other in PND, in order to maximize the diagnosis rate and reduce the occurrence of birth defects.


| INTRODUCTION
Prenatal diagnosis (PND) is a technique employed to detect the chromosomal abnormalities in fetuses before birth, which is an important means of reducing birth defects (Alesi, Bertoli, Sinibaldi, & Novelli, 2013). Karyotyping is the gold standard for PND and can detect aneuploidies and large structural chromosome rearrangements (>5 Mb) . With the advent of molecular cytogenetic technologies such as fluorescence in situ hybridization (FISH) and chromosome microarray analysis (CMA), it is now understood that chromosomal microdeletions and microduplications below the karyotyping resolution contribute significantly to diseases (Cui et al., 2019;Quintela et al., 2015;Yi et al., 2019). The application of various molecular diagnostic techniques to detect microdeletions and microduplications plays a critical role in PND (Chen et al., 2014;Karcaaltincaba et al., 2010;Klugman et al., 2014). BACs-on-Beads (BoBs™) assay was modified from comparative genomic hybridization and developed to detect the DNA copy number gains and losses. BoBs™ assay is a bead-based multiple assay using beads impregnated with two different fluorochromes of unlike concentrations to create an array of up to 100 different unique probes, each probe is derived from a BAC DNA that can allow the diagnosis of common abnormalities and nine microdeletions within 30 h (García-Herrero et al., 2014). Here, we report that BoBs™ assay has unique application advantages in PND.

| Ethical compliance
The study design and protocol were reviewed and approved by the ethics committee of Fujian Maternity and Child Health Hospital (No. 2016021). All procedures performed in these studies involving human participants were conducted in accordance with the ethical standards of the institutional and/ or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

| Study design
1520 samples were collected between July 2017 and June 2019 in Fujian Maternity and Child Health Hospital (Fujian, China) and included seven chorionic villi biopsy samples (7/1520,0.46%), 1328 amniotic fluid samples (1328/1520,87.37%), and 185 umbilical cord centesis samples (185/1520, 12.17%). Pregnant women from 18 to 43 years of age with an average age of 24.63 ± 2.47 years were considered. According to the clinical indications for invasive PND, detailed characteristic of these gravid women are displayed in Table 1, including simple advanced age (n = 506), high risk of NIPT (n = 31), high-risk pregnancy of serological screening in early and middle stages (n = 315), abnormal ultrasound (n = 398), adverse pregnancy history (n = 35), two kinds of abnormal indications (n = 198), three kinds of abnormal indications (n = 19), and others categories (n = 18) assessed in this study.

| DNA isolation
Genomic DNA was extracted using a commercially available DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The extracted DNA was quantified by a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA). Finally, the genomic DNA was stored at −80°C until further use.

| BoBs ™ assay
BoBs™ assay can detect the copy number changes of chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes. The nine microdeletion syndromes analyzed were Wolf-Hirschhorn syndrome ( Table 2. This assay was obtained from BoBs™ assay manufacturer (PerkinElmer, Wallac Oy, Finland), and the fluorescence data were analyzed with BoBsoft software (PerkinElmer, Wallac Oy, Finland). The samples were defined as deleted/ duplicated at a specific chromosomal locus when the ratios of the fluorescence intensities fell outside the threshold of the mean ± 2 SDs, typically ranging between 0.6 and 0.8 (deleted) and between 1.3 and 1.4 (duplicated), respectively .

| Chromosome microarray analysis
The microdeletions or microduplications detected by BoBs™ assay were then validated with CMA using the Thermo Fisher CytoScan 750 K arrays (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocols. Data were analyzed using Chromosome Analysis Suite software 3.1.

| Fluorescence in situ hybridization
FISH assay was performed on one case using an AneuVysion Assay Kit (Abbott Park, Illinois, USA). The D18Z1, DXZ1, and DYZ3 probes were selected based on the gain region detected by CMA. Metaphase FISH analysis was performed on the amniocytes according to the manufacturer's protocol.

| Statistical analysis
Statistical analyses were performed using SPSS 18.0 (IBM, USA). Data were presented as the mean ± SD (standard deviation) between three independent experiments with each being measured in triplicate. The differences among the groups were analyzed using One-way ANOVA. A value of p < 0.05 was considered being a statistically significant difference.

| Mosaicism
Four cases of mosaicism were identified by both prenatal BoBs™ and karyotyping. One case with small supernumerary marker chromosomes was verified by CMA and FISH (Table 5). Two cases with small supernumerary marker chromosomes were verified by CMA only. BoBs™, CMA, and FISH maps of mosaicism are shown in Figure 4.

| Pregnancy outcomes
Of the 1520 prenatal cases collected, 1428 cases were successfully followed up, while 92 cases were lost. The followup rate was 93.95% (1428/1520). Of the 76 abnormal cases detected, 74 cases were terminated and two had normal deliveries. Among 1352 cases with normal chromosomes, 79 cases were terminated due to obvious abnormalities in ultrasound, and 1273 cases had normal deliveries.

| DISCUSSION
Chromosomal abnormality is the main reason for congenital anomalies. It is estimated that six percent of congenital defects are due to aneuploidies and nearly one in 200 newborns is affected. Karyotyping is routinely used for PND of chromosomal abnormalities such as aneuploidy and balanced translocations. However, it cannot detect deletions and duplications of small fragments of less than 5 Mb . In addition, it has many disadvantages, including the need for cell culture, greater manual operation time, long reporting cycles (the average reporting time is 14 days), and the possibility of cultivating failure. Prenatal BoBs™ technology can detect most common aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes found at high incidence in human population (the accumulative incidence can reach 1/1700) (Shaffer & Van den Veyver, 2012) without cell culture. Furthermore, BoBs™ assay has the advantage of short detection period (it can be completed within 30 h), simple operation procedure, high screening rate (92 samples can be  (Vialard et al., 2011). Therefore, we should balance the benefits and drawbacks of conventional karyotyping against BoBs™ assay.
The results of our retrospective study on 1520 samples from Fujian province in China have provided further assurance on the increased diagnostic yield of BoBs™ assay compared with karyotyping. The chorionic villi biopsy, amniotic fluid, and umbilical cord centesis samples were cultured, and conventional karyotyping was performed in parallel. The success rate of BoBs™ assay was higher than that of karyotyping (100% vs. 98.09%). Around 27 cases of Trisomy 21, 11 cases of Trisomy 18, 4 cases of Trisomy 13, and 17 cases of sex chromosomal aneuploidy were detected by both karyotyping and BoBs™ assay. BoBs™ assay detected one case of WHS syndrome that was verified by CMA and was consistent with karyotyping results. The missing fragments above were 35 MB, covering all areas of the syndrome. Based on our data, the application of BoBs™ assay in PND is very trustworthy and advocating involved multicenter research (Vialard et al., 2011. BoBs™ assay also detected three cases of DGS microdeletions, two cases of MDS microdeletions, one case of SMS microdeletions, one case of WBS microdeletions, one case of PWS microdeletions, one case of Xp22.31 Microdeletions, one case of 22q11.2 microduplications, and one case of 17p11.2 microduplications, whereas karyotyping failed to reveal these chromosomal abnormalities. Furthermore, all these chromosomal abnormalities were verified by CMA. Microduplications were not the scope of the BoBs™ report, but the results can also have a certain role in suggesting the involvement of a microduplication syndrome, while additional prompts can improve detection rates. Submicroscopic deletions at Xp22.31 lead to the loss of STS (MIM: 300747) and ANOS1 (MIM: 300836) genes, which can cause X-linked ichthyosis and Kallmann syndrome, respectively (Nagai et al., 2017;Quintela et al., 2015). Male patients with X-linked ichthyosis also exhibits severe hypogenitalism and hypogonadism (Nagai et al., 2017). The case with Xp22.31 microdeletions detected in the study was a female fetus carrier of ichthyosis without skin abnormalities followed up at 6 months post-partum. The chromosome 22q11.2 deletion has been implicated in genomic diseases for long (Stachon et al., 2007). Chromosome duplications of the region in patients have been reported, establishing a new genomic duplication syndrome (Hoeffding et al., 2017;Li, Yi, et al., 2019;Portnoi, 2009;Vaisvilas et al., 2018) complementary to the 22q11.2 deletion syndrome. In the study, one fetus with 22q11.2 microduplications was identified who showed ventricular defect. The pregnancy was terminated. Potocki-Lupski syndrome (PTLS) occurs in approximately 1 in 25,000 births and is associated with congenital anomalies and intellectual disability (Popowski et al., 2012). PTLS is caused by genetic duplication within 17p11.2 region (Potocki et al., 2000(Potocki et al., , 2007. The fetus with 17p11.2 microduplications detected in the study had cardiovascular abnormalities and similarly the pregnancy was terminated. As long as the area covered by BoBs™ probe can be detected, BoBs™ assay can offer additional diagnostic benefit compared with karyotyping and provide greater sensitivity for detecting microdeletions and microduplications (García-Herrero et al., 2014;Gross et al., 2011).
One case involving 8q21.2q23.3 duplication was found by karyotyping but was not detected by BoBs™ assay. CMA showed an increased copy number of the pathogenic genome in chromosome 8q21.2q23.3 with copy number three and a fragment size of about 28 Mb. This increased region contains multiple pathogenic OMIM genes that can cause growth retardation, mental retardation, craniofacial deformities, multiple malformations, and congenital anomalies such as heart and kidney malformations (Piotrowski et al., 2014;Tsang, Yang, & Fong, 2014). The fetus with 8q21.2q23.3 duplication had cardiovascular abnormalities and the pregnancy was terminated. Since the added area was not within the BoBs™ probe coverage, the result of BoBs™ assay was normal. The limited detection range of BoBs™ assay reduces the detection of abnormal rate. Mosaicism was observed in 0.8%-1.5% of prenatal samples (Hoang et al., 2011). At present, the most commonly used method to assess mosaicism is karyotyping. Its accuracy is depended not only on the proportion of abnormal cells present but also on the number of cells being analyzed (Chen et al., 2016). The ability of prenatal BoBs™ assay to detect chromosome mosaicism has been explored with a 20% to 30% ratio of mosaicism (Cheng et al., 2013). In our study, four cases of mosaicism at different ratios were detected, the lowest of which was 14%. This showed that the ability of BoBs™ assay to detect chromosome mosaicism was beyond our previous expectations. For those three cases of sex chromosome mosaicism, BoBs™ assay was only able to detect sex chromosome abnormality. Karyotype analysis can only determine the ratio of mosaicism, while CMA and FISH can clarify the sources of small supernumerary marker chromosomes. Of the three small marker chromosomes, two were parts of X chromosome and one was part of Y. The combined analysis of multiple detection methods can better explain the results. BoBs™ assay and CMA were based on uncultured cells, which can reflect the fetal situation more realistically. PND of 45,X/46,XY mosaicism occurs approximately in 1.7 per 10,000 prenatal samples with phenotype ranging from 90% of normal male fetuses to postnatal features that include a wide spectrum of phenotypes such as hypospadias (Barone et al., 2011). PND plays an important role in preventing the transmission of genetic abnormalities. All the cases of mosaicism in our study informed choices to terminate pregnancies.
Based on our data, the anomaly rate of simple advanced age, high risk of NIPT, high-risk pregnancy of serological screening, abnormal ultrasound, adverse pregnancy history, and other factors was not significantly different (p > 0.05). PND of all high-risk pregnant women is very necessary.
Our study exhibited some limitations. Samples with common aneuploidies were not detected by CMA as improving test results. The study lacked multicenter collaboration and large amount of data. Thus, further studies with multicenter surveys and larger sample sizes are required to confirm our findings.
In conclusion, BoBs™ assay is considered a reliable, rapid test for PND. A variety of comprehensive technological applications can complement one another in PND in order to maximize the diagnosis rate and reduce the occurrence of birth defects.