It is well known that the role of copy number variants (CNVs) has become increasingly apparent. Researchers have found that CNVs have been linked to numerous variety of human diseases, including intellectual disability (ID), multiple congenital anomaly syndromes, and complex neurodegenerative and neuropsychiatric disorders [1, 2]. Structural genomic rearrangements such as duplications, deletions, translocations, and inversions were the major cause of CNVs [3, 4]. If a parent carries the structural abnormalities of chromosome balance including balanced translocation, Robertsonian translocation or inversion without any loss or gain of genetic material, it is likely to result in CNVs in the offspring [5, 6].

With the rapid development of chromosomal microarray technology, few complex CNVs (number of CNV $\ge$2) cases have been reported over the last decade. Researchers hypothesized that the complex CNVs were associated with more severe clinical expressivity of known syndromes. Bertini et al. [7] suggested that additional CNVs outside the 22q11.2 region may possibly modulate the variable phenotype and incomplete penetrance of DiGeorge syndrome, as well as proposed “Additional Hit” to explain severe expressivity [8]. The majority of 22q cases are de novo (93%) and are usually a result of non allelic homologus recombination [9]. The most common known balanced translocation in chromosomes 11 and 22 increase the risk of an unbalanced gamete with 22q11 (up to 6% risk) [9]. Therefore, it is important that parental study and genetic counseling be performed in prenatal cases with complex CNVs.

The objective of this report is to describe 12 cases of complex fetal CNVs diagnosed by prenatal chromosomal microarray. We will also review the available parental studies, including use of karyotype, chromosomal microarray analysis (CMA), and whole-genome low-coverage mate-pair sequencing (WGL-MPS) to better clarify the complex fetal rearrangement.

Of the 2746 cases diagnosed with CMA, fetuses that carried complex CNVs were identified in 0.4% of cases (12/2746). Clinical information and microarray findings of 12 cases are summarized in Table 1.

Among the 12 cases, gestational age of patients were between 15 and 26 (21.3 $\pm$ 3.6) weeks and maternal age were from 24 to 35 (29.1 $\pm$ 3.4) years old. Clinical indications included ultrasound anomaly, abnormal pregnancy history and abnormal prenatal screening. These results were recorded as fetal growth restriction (FGR), congenital heart disease (CHD), screening for Down’s syndrome (DS) ratio or other high-risk indicators of chromosomal anomaly. Ninety two percent (11/12) of cases carried two complex CNVs, and only Case 10 had co-occurrence of 3 complex CNVs. CNVs of all cases were classified as P and VUS according to the ACMG guideline and relevant database. In accordance with CMA findings and clinical indications, all patients voluntarily chose termination of pregnancy after prenatal genetic counseling and they do not have the pregnancy plan at the end of our 2-year follow up. Unfortunately, all families refused autopsy due to Chinese taboo.

The parental validation study was carried out in 8 of 12 complex CNVs cases with the permission of the patients, while others have declined the study. All study details are shown in Table 2. On the basis of different CNVs sizes, which were from 680 Kb to 21.9 Mb, parental specimens were performed by different validation methods. G banding karyotype (Case 1, 2, 7, 8, 9, 11, and 12), CMA (Case 10), and WGL-MPS analysis (Case 8 and 12) were selected depending on the resolution of methods and CNVs sizes to determine the origin of the variants. Case 8 and 12 were analyzed by karyotype and then validated by WGL-MPS analysis due to the relatively small imbalance size. The primary results suggested that 62.5% (5 out of 8) of fetuses carried complex CNVs that were inherited, of which 4 cases were derived from maternal or paternal balanced translocation carriers. Miscarriage history was found in some of the balanced translocation carriers (Case 1, 2 and 11).

According to Tables 1,2, Case 0 showed a rare complex CNVs with three microduplications and microdeletions that were all inherited from her healthy parents. Considering the size of loss and gain, this case was validated using CMA directly (seen in Fig. 1).

Fig. 1.

Chromosomal microarray CNV log2 ratio profile of Case 10. (a) 15q11.2 deletion region profile of the proband and his mother. (b) 18q22.2 duplication imbalance of the proband and his mother. (c) 16p13.11 deletion region profile of the proband and his father. CNV, copy number variant.

The proband was diagnosed with 15q11.2 BP1-BP2 microdeletion (Burnside-Butler) syndrome with incomplete and highly variable penetrance at around 10.4% [12], with typical phenotypes including neurodevelopmental disorders with changes in cognition and behavior, 16p13.11 microdeletion syndrome and a VUS CNV duplication at 18q12.2. The proband’s mother carried the 15q11.2 deletion involving four OMIM genes (TUBGCP5, CYFIP1, NIPA2, NIPA1) and 18q12.2 duplication including 6 OMIM genes (MIR187, RPRD1A, SLC39A, ELP2, MOCOS, FHOD3). She did not have a family history of intellectual disability or developmental delay. Copy number variant on 16p13.11 region with incomplete penetrance and highly variable phenotypic manifestations, which were 1.6 Mb encompassing 11 OMIM genes (i.e., NED1), were inherited from the phenotypically normal father of the proband. These parents choose to terminate the pregnancy after genetic counseling.

In this study, we described 12 prenatal coexisting CNVs cases diagnosed by CMA, 8 of which were followed by parental validation analysis performed via different genetic approaches. Sixty-two and one half percent (5/8) of complex CNVs were inherited and 80% (4/5) of these cases found in balanced translocation carriers. Under these circumstances, CNVs inheritance mode (paternal/maternal or de novo) can have a serious impact on genetic counseling for the recurrence risk in future pregnancies of families experiencing complex CNVs [8].

Coexisting CNVs cases were not common during in the past [9]. From 2017 to 2020, complex CNVs (co-occurrence of two or more) in one fetus were a rare phenomenon in our local prenatal CMA cohort, accounting for 0.4%. Previous studies have shown that coexisting abnormalities can occur in the context of complex CNVs (Table 3, Ref. [7, 8, 9, 10, 13, 14, 15, 16]).

Regarding prenatal diagnosis and complex CNVs interpretation, CNVs origin can have an impact on patients and their families. In this study, half of the inherited complex CNVs cases were derived from a balanced translocation in the parents. In the general population, the percentage of balanced translocation carriers is 0.08~0.3 [17]. Balanced translocation carriers have an increased risk for recurrent miscarriage, infertility, or birth of a child with CNVs [18, 19]. Therefore, it is necessary for the genetic counselor assist the parents in understanding the genetic information if the family has a diagnosis of fetal complex CNVs.

Genetic counseling can have a significant impact on the pregnancy by providing information, support, and guidance to expectant parents [20]. Meanwhile, the genetics counselor is able to assist families to cope with the stress and uncertainty that can accompany a diagnosis of a fetal anomaly or complex CNVs, and then provide resources or support to promote positive mental health outcomes for the parents [21]. Moreover, in cases where a fetal anomaly is detected, genetic counselors can help parents understand the implications of the diagnosis and provide support as they make decisions about how to proceed. This may include decisions about whether to continue or terminate the pregnancy, as well as decisions about future family planning [20].

In our study, counseling was performed on all patients based on their CMA results and parental analysis. For de novo cases, the risk of recurrence of these complex imbalances is estimated at 1% according to the literature (including germinal mosaicism), which is reassuring for patients considering their next pregnancy [22]. Likewise, prenatal diagnosis and counseling are highly recommended for their next pregnancy. Considering cases carrying complex CNVs that are transmitted by their balanced translocation parents, the risk of imbalances recurruring is relatively higher than in de novo cases and it is more difficult for carriers to have a healthy baby due to repeated spontaneous abortion and imbalance occurrence in future pregnancies [22]. Therefore, genetic counseling has a significant role for these families’ future pregnancies. For carriers who are willing to have further attempts for natural conception, early invasive prenatal diagnosis might be a choice for their pregnancy during the first trimester. For patients who have experienced miscarriages, infertility, and birth of sick children, as well as having adequate financial ability, preimplantation genetic diagnosis (PGD) is another option to increase the likelihood of successful pregnancy and decrease the risk of a genetic disorder in their offspring [23].

However, the limitations of this cohort study should be noted. Even though this research has a large cohort with 2746 pregnant patients with complete fetal data and whole genome parental studies for accurate parental genotyping, the number of complex CNVs cases were relatively small. Furthermore, other limitations are the loss of follow up of 4/12 parents, no data from patients about pregnancy experience or impact of genetic counseling and lack of the follow up on future pregnancies of proband families. In the future, we will expand the number of cases and extend the time of data collection with more CNVs origin analysis, making more efforts to initiate complex CNVs counseling and follow-up studies on patients.

In conclusion, we used CMA technology to diagnose complex CNVs in fetuses followed by parental analysis via different validation methods according to various resolutions and sizes of CNVs. Complex CNVs cases were rare in our cohort (0.4%) as well as in previous reported studies (0.9%) [8, 12, 13]. In these cases, parental analysis can vastly change the recurrence risk for patients. For our study, the CNVs origin analysis demonstrated that the inherited complex CNVs account for 62.5% and 80% of inherited cases were transmitted from balanced translocation parents. Based on the complexity of CNVs cases and the wide range of possible recurrence risks, these patients will benefit from prenatal genetic counseling. Prenatal counseling can assist patients to make informed decisions about their current and future pregnancies.

The data used to support the findings of this study are available from the corresponding authors upon request.

WW, JW and BZ designed the research study. WW and JW performed the research. YS and BZ provided help and advice on the CMA and parental study experiments. WW and JW analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study design and protocol were reviewed and approved by the ethics committee of Changzhou Maternal and Child Health Care Hospital (Approval number: 2017003). Written informed contents were obtained from the patients before screening. All patients signed written informed consent for publication of their clinical data.

We are thankful to the patient and the family for their participation in this study.

This study was funded by National Natural Science Foundation Youth Fund (No. 82103853), Changzhou science and technology support project (Social development) (CE20225066) and Top Talent of Changzhou “The 14th Five-Year Plan” High-Level Health Talents Training Project (2022CZBJ089).

The authors declare no conflict of interest.