Combined use of karyotyping and copy number variation sequencing technology in prenatal diagnosis

Introduction

Karyotyping of amniotic fluid cells is still the most common technique used to identify chromosomal abnormalities and has been the gold standard in prenatal cytogenetic analysis. However, due to its long detection period and low detection resolution, it is unable to identify genomic copy number variations (CNVs) smaller than 10 Mb. CNVs are losses or gains of genomic segments and are a type of structural variation. CNVs are usually defined as genomic segments and present variable copy numbers that are 1 Kb or larger when compared with a reference genome (Nevado et al., 2014). A chromosomal microarray (CMA) is mainly used for the detection of genome-wide CNVs and plays a very significant role in the prenatal and postnatal samples for the detection of chromosomal aberrations (Cheng et al., 2019; Wang et al., 2020). However, due to its high cost, low throughput, and complex experimental procedures, the large-scale application of this technique in prenatal diagnosis is limited. Moreover, the limited coverage of the CMA probe presents the possibility that some pathogenic copy number variations (pCNVs) may not be detected (Dong et al., 2016; Hayes et al., 2013).

Based on the emergence of next-generation sequencing (NGS) technology, the detection of CNVs has a wider range, higher throughput, lower cost, shorter reporting period, and lower DNA sample requirements, making it more suitable for clinical applications (Liang et al., 2014; Liu et al., 2015; Xie & Tammi, 2009; Zhang et al., 2021b; Zhu et al., 2016). Therefore, we speculated that a simultaneous analysis and comparison of the results from karyotyping and genome copy number variation sequencing (CNV-seq) may be more effective in the diagnosis of chromosomal abnormalities and improve the accuracy of diagnosis. To explore this theory, we explore the possibility of using a combination of the two methods in the prenatal diagnosis of chromosomal abnormalities in 822 pregnant women who underwent traditional karyotype analysis and CNV-seq testing simultaneously.

Patients & Methods

Results

Results of two special cases

In this study, the chromosomes of cases 22 and 39 were special. The prenatal diagnosis indication of case 22 was NT 3.8 mm, and the karyotype was 45, XN, rob (14; 21) (q10; q10). The translocation of chromosome 21 is larger than that of normal chromosome 21, and it is difficult to judge whether it is abnormal karyotype and the source of the abnormal fragment. Case 22 was confirmed as seq[GRCh38]dup(21)(q21.1q22.3)(19047682-46680088) × 3, which showed 27.68 Mb duplication in the 21q21.1q22.3 region and was consistent with trisomy 21. The prenatal diagnosis indicator of case 39 was maternal serum screening high risk (1/61), and the karyotype was 46, XN. However, case 39 was confirmed as seq[GRCh38]del(5)(p15.33p15.1) (20001-17939891) × 1, and seq[GRCh38]dup(7)(q34q36.3) (141680201-159335973) × 3, which showed 17.92 Mb deletion in the 5p15.33p15.1 region, and 17.76 Mb duplication in the 7q34q36.3 region. The results of karyotypes and CNV-seq of case 22 and 39 are shown in Figs. 1 and 2, respectively. Therefore, we concluded that CNV-seq could be used as an effective complement to karyotyping.

Discussion

Today, abnormality of fetal chromosome number or structure is the main cause of fetal malformation, abortion, stillbirth, and neonatal death. Traditional cytogenetic analysis is quite limited when looking for submicroscopic chromosomal changes, namely CNVs. So far, over 300 kinds of chromosome microdeletion/ duplication syndromes caused by pCNVs have been identified, with an incidence rate of 1/600 (Goldenberg, 2018; Nevado et al., 2014), accounting for half of the birth defects caused by chromosomal aberrations (Evans, Wapner & Berkowitz, 2016). Previous studies have shown that 6–7% of fetuses with normal karyotypes but abnormal structure indicated by ultrasound have definite or possible pathogenic CNVs (Callaway et al., 2013; Hillman et al., 2013; Wapner et al., 2012). Therefore, a combination of karyotyping and CNV-seq has gradually been used in prenatal diagnosis.

In this study, 822 women underwent traditional karyotype analysis and CNV-seq test. The highest constituent ratio of amniocentesis indicators was in abnormal fetal ultrasound (39.29%), those patients with abnormal ultrasound were more inclined to choose the combined detection of the two technologies (Wang et al., 2018; Zhang et al., 2021a; Zhao & Fu, 2019). Thus, abnormal fetal ultrasonography is a major indicator for prenatal molecular diagnosis. The abnormal detection rate was 13.93% in the group with prenatal ultrasound abnormalities, which was similar to the research results of Wang et al. (2018).

We found that the abnormal detection rate of the NIPT high-risk group was significantly higher than all of the other groups, and the abnormal detection rate of mixed indicators was significantly higher than the history of the adverse reproductive outcomes group. NIPT is highly accurate and has been effectively and widely used as a prenatal non-invasive screening method (Zhang et al., 2015). In addition to trisomy 21, 18, and 13 routine screening, NIPT has a certain detection effect on sex chromosome aneuploidy and fetal pathogenic microdeletion/duplication, and can provide clinical application value for subsequent prenatal diagnosis. However, since NIPT is also a sequencing technology platform, it has the same defects as CNV-seq technology and cannot detect fetal polymorphism, balanced translocations, polyploids, and other fetal structural abnormalities. The chromosome abnormal detection rate of a fetus in the mixed group of multiple indicators was higher, this was because some people in the group were carriers of chromosome balanced translocation.

In the present study, the abnormal detection rate of combination of two or more indicators group was not significantly higher than that of single indicator group. Previous study found that multiple prenatal diagnosis indicators could decrease the sensitivity but increase the specificity to predict fetal pathogenic CNV (Zhang et al., 2021b). We acknowledged that there were some limitations of this study including relatively small sample size and its retrospective nature, which may predispose the study to selection bias and issues with missing data. Therefore, possible prenatal predictive efficiencies of combined different indicators for pathogenic chromosomal abnormalities required additional investigation.

In general, these results and Chinese expert consensus on the application of low-depth whole genome sequencing in prenatal diagnosis (Clinical Genetics Group of Medical Genetics Branch Chinese Medical et al., 2019) suggest that karyotyping and CNV-seq could be recommended as first-line prenatal diagnosis methods for pregnant women with the six high-risk indicators.

Combination of CNV-seq and karyotyping significantly could improve the detection rate of fetal pathogenic chromosomal abnormalities. Two combined detection methods found a total of 119 abnormal cases, which made up 14.48% of the total. There were 57 cases of chromosomal abnormalities that were detected by karyotyping, accounting for 6.93% of the total subjects. There were 99 women who were confirmed to have chromosomal abnormalities (pathogenic, likely pathogenic, and VUS) that were detected by CNV-seq, accounting for 12.04% of the total subjects. Among these patients, the abnormal detection rate for the pathogenic and likely pathogenic variants was 8.03% (66/822). Compared with karyotyping, the abnormal detection rate of pathogenic/likely pathogenic CNV-seq was increased by 1.10%, which was similar to the results of other studies (Wang et al., 2019; Wang et al., 2018).

CNV-seq could accurately locate the abnormal fragments of karyotypes and provide more accurate genetic information during prenatal diagnosis and clinical genetic counseling. Our research results showed that CNV-seq could detect all chromosomal aneuploidy abnormalities, such as trisomy 21, trisomy 18, and sex chromosome abnormalities. The results of CNV-seq and karyotyping were consistent. Notably, case 22 had Robertsonian translocation between chromosomes 14 and 21, which is the most common Rb translocation found in humans. CNV-seq analysis showed a case of 21q21.1-q22.3 duplication (27.68 Mb), which is the key region in Down’s syndrome. In case 29, it was impossible to analyze Y chromosomes or small supernumerary marker chromosomes (sSMC) using karyotype analysis. However, CNV-seq suggested that the Y chromosome was amphiploid, thus the clinical diagnosis was 47, XYY. In case 32, there was a balanced translocation between chromosome 4 and chromosome 16 in the father of the fetus. Chromosome balanced translocation carriers are prone to produce unbalanced gametes (Morin et al., 2017; Wang et al., 2016), such as in this case of 16q21-q24.3 duplication (27.18 Mb) and 4q35.1-q35.2 deletion (6.52 Mb). The clinical phenotype obtained from the database query was consistent with that of fetal congenital heart disease (double outlet right ventricle, ventricular defect) indicated by B-ultrasound, which suggested that these were clinically relevant CNVs. NIPT in case 33 suggested that other chromosomal abnormalities might occur in the fetus. CNV-seq showed 8q22.1q24.3 duplication (48.5 Mb) and 12q24.33 deletion (1.1 Mb). The duplication region of 8q22.1q24.3 belongs to trisomy 8 (including the key segment of 8q22-q24). The clinical features include short stature, special facial features, cryptorchidism, hypertrichosis, congenital heart disease, intellectual disability, and frequent seizures. The CNVs were also clinically relevant.

CNV-seq can not only accurately locate the source of abnormal chromosomal fragments, but also find chromosomal microdeletions and microduplications that cannot be found by karyotyping. In this study, we found 22 cases of pathogenic CNVs, eight cases of likely pathogenic CNVs, and 33 cases of VUS that could not be detected by karyotyping. Among the 22 cases of pathogenic CNVs, cases 40 and 48 were the results of two pregnancies from the same woman, and the fetus was retained after informed consent of the second pregnancy. The clinical manifestations of this pregnant woman were mainly special facial features, skeletal dysplasia, and intellectual disability. The genotype of the mother was seq[GRCh38]del(1)(p36.33p36.32) NC_000001.11:g.884621_2823435del. The two fetal pathogenic CNVs inherited from the mother of the fetus could cause 1p36 microdeletion syndrome. It is worth mentioning that case 39 showed 17.92 Mb deletion in the 5p15.33p15.1 region (containing Cri du chat syndrome key genes), and 17.76 Mb duplication in the 7q34q36.3 region. Since karyotyping results do not indicate cytogenetic abnormalities, a confirmatory test such as FISH needs to be performed following CNV-seq. However, this pregnant woman refused to undergo FISH testing and chose to terminate her pregnancy voluntarily after genetic counseling.

Why did karyotype analysis not detect the chromosome abnormalities larger than 10 Mb? Due to the morphologic similarities between 5p15.33p15.1 and 7q34q36.3, it was difficult for karyotype analysis to accurately distinguish between subtle structural variations. We speculated that the short arm end of chromosome 5 was actually 7q34q36.3 translocation, and the two chromosomes 7 were normal.

Additionally, we found that the proportion of VUS microdeletion/duplication was 4.01% (33/822), which was slightly higher the results of Wang et al. (2018). VUS presents challenges to clinical genetic counseling. Clinical intervention should be combined with the clinical phenotype and penetrance of CNV. Parental DNA testing by CNV-seq can help further interpret the pathogenicity of the fetal CNVs and define parental origin, so that the information could be used by the clinician to help interpret these VUS results, and manage these aneuploid pregnancies. In addition, most of the investigated VUS were proven to be de novo (Wang et al., 2018). For the clinicians and patients, the discovery of a de novo VUS is problematic and follow-up after birth is recommended by the ACMG (Richards et al., 2015). In this study, we explored the pathogenicity of microdeletion/duplication in chromosome Xp22.31.

Cases 56–58 were male fetuses with deletion of 1.68 Mb regions on chromosome Xp22.31, which contains the entire steroid sulfatase gene (STS). Mutations and partial or entire deletions of STS have been reported to cause XLI (Zhang et al., 2020). The main clinical manifestation of XLI is large areas of scales on the limbs, face, neck, trunk, and buttocks. These skin lesions persist and do not improve with age. XLI occurs almost only in male patients at birth or shortly after birth. Due to the influence of potential factors such as X chromosome abnormal fragment size, connection position, random inactivation, bias inactivation, and gene escape inactivation, female carriers of X chromosome abnormal fragments may have some clinical variability. In our study, two cases were inherited from the maternal side and one case had familial ichthyosis. After genetic counseling, three pregnant women chose to continue the pregnancy with informed consent.

The fetuses of cases 91-96 had Xp22.31 duplications ranging from 1.5 Kb to 1.7 Mb. This region covered four genes: PUDP, STS, VCX, and PNPLA4. The pathogenicity of Xp22.31 duplication seems to be controversial. Previous reports have shown that some individuals with duplication of this region had varied degrees of neurological impairment, including growth retardation, intellectual disability, autistic spectrum disorders, hypotonia, seizures, psychomotor retardation, and mild special face (Faletra et al., 2012; Pavone et al., 2019; Polo-Antunez & Arroyo-Carrera, 2017). Some studies showed that duplication of Xp22.31 is a risk factor for abnormal phenotypes or benign variants (Liu et al., 2011; Zhuang et al., 2019). In this study, we found that five babies with Xp22.31 duplication did not present with phenotypes during follow-up. We will continue to follow up on these cases to observe if they may show clinical phenotypes consistent with the disease-causing genes as they age. Therefore, the duplications of Xp22.31 with recurrent duplication may serve as VUS.

CNV-seq could not detect the balanced translocation and inversion of chromosomes, which can be detected by karyotyping, but CNV-seq can detect whether the balanced translocation is accompanied by chromosome microdeletion/ duplication. The CNV-seq results of the 20 cases (Table S4) were normal, confirming that CNV-seq could not detect the balanced translocation and inversion of chromosomes, and there was no increase or decrease of pathogenic genetic material during chromosome rearrangement (Cohen et al., 2015; Zhao & Fu, 2019). Balanced translocations and inversions of chromosomes are important causes of reproductive abnormalities. Couples in whom one partner has a balanced translocation or inversion may have an overall high miscarriage rate resulting from unbalanced gametes (Kaser, 2018). Due to the high possibility of abnormal gametes, the risk of recurrent abortion and birth of children with abnormal chromosomes also increased. It is suggested that the appropriate fertility program be selected according to the specific situation, such as prenatal diagnosis after natural pregnancy or the use of preimplantation genetic diagnosis (PGD) technology to select normal embryo transfer (Liu et al., 2015).

CNV-seq could verify pathogenicity of sSMC in prenatal diagnosis. sSMC, also known as marker chromosome or extra abnormal structure chromosome, refers to the redundant chromosome with morphology that can be identified, but its characteristics and source cannot be identified by traditional karyotyping technology (Mcgowan-Jordan, Hastings & Moore, 2020). In order to accurately determine the clinical phenotype and survival of the fetus, it was necessary to detect the fetuses with sSMC by cytogenetic and molecular methods. Previous studies have reported that the detection rate of fetal sSMC in prenatal diagnoses was 0.8–1.51‰ (Huang et al., 2012; Huang et al., 2019). In this study, only one sSMC was found in 2631 cases of amniocentesis by karyotyping. The CNV-seq results showed that it did not contain known human disease-related pathogenic genes, which presumably did not increase the risk of sub-representational abnormalities, and the patient opted to continue the pregnancy following clinical counseling.

Conclusion

In conclusion, karyotyping and CNV-seq have their own advantages and disadvantages in prenatal diagnosis. Using a combination of CNV-seq and karyotyping significantly improved the detection rate of fetal pathogenic chromosomal abnormalities. CNV-seq is an effective complement to karyotyping and improves the accuracy of prenatal diagnosis. NIPT is a recommended non-invasive prenatal screening method for fetal chromosomal abnormalities. It is believed that with the widespread application of CNV-seq, the pathogenicity of more VUS microdeletion/duplication will be explored, as will the development of clinical genetic counseling.

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