Identification of Complex Chromosomal Rearrangement Involving Chromosomes 10, 18, and 19 in a Family Undergoing Prenatal Diagnosis: Case Report

ABSTRACT

Complex chromosomal rearrangements (CCRs) are rare chromosomal structural abnormalities that are often difficult to be detected. Chromosome conformation‐based karyotyping (c‐Moka) is an emerging technology designed to analyze the three‐dimensional structure of chromosomes, thereby identifying various types of chromosomal structural variations. In our study, conventional karyotyping was performed for a gravida with a high risk of sex chromosome abnormality, as indicated by non‐invasive prenatal testing. The analysis suggested a potential chromosome translocation at the terminals of chromosomes 18 and 19. Subsequently, fluorescence in situ hybridization (FISH) was performed at metaphase stage using a 19p13.3 probe. The signal was detected on other chromosome rather than chromosome 18, leading us to hypothesize that the subject might have CCRs that was not identifiable through conventional karyotyping and FISH. To further investigate, we employed c‐Moka, which revealed CCRs involving chromosomes 10, 18 and 19. Ultimately, the karyotype of the subject was determined to be 46,XX,t(10;18;19)(q26.3;p11.21;p13.3). This study underscores that, compared to conventional karyotyping, c‐Moka has significant advantages in detecting CCRs.

Key Clinical Message

Complex chromosomal rearrangements (CCRs) are rare cytogenetic findings. This report documents the first case in which chromosome conformation‐based karyotyping (c‐Moka) revealed CCRs involving chromosomes 10, 18 and 19, which were misdiagnosed by conventional karyotyping and highlights the significant advantages of c‐Moka in detecting CCRs.

1. Introduction

Complex chromosomal rearrangements (CCRs) are rare cytogenetic findings characterized by balanced or unbalanced rearrangements involving at least three chromosomes and three or more cytogenetic breakpoints [1]. Patients with CCRs frequently present with intellectual disability, multiple congenital anomalies, infertility, or recurrent pregnancy loss. The proposed mechanisms underlying CCRs include the Fork Stalling and Template Switching (FoSTeS) models and microhomology‐mediated break‐induced replication (MMBIR).

Traditional methods for detecting CCRs include conventional karyotyping, chromosomal microarray analysis (CMA), copy number variation (CNV), fluorescent in situ hybridization (FISH), and multicolour fluorescence in situ hybridization (M‐FISH) [2]. However, each of these approaches has inherent limitations. Conventional karyotyping can only resolve chromosomal structural changes larger than 5–10 Mb, making it difficult to detect the cryptic fragments smaller than 5 Mb at the gene level. CMA and CNV analysis are unable to identify balanced CCRs. Although FISH and M‐FISH offer high sensitivity through the use of specific probes, their applicability is constrained by probe availability, which results in the misdiagnosis of cryptic CCRs.

c‐MoKa is an innovative technology that integrates chromatin conformation capture with high‐throughput sequencing [3]. This technique involves crosslinking DNA with proteins through formaldehyde treatment to fix the DNA conformation, followed by enzyme digestion, biotin introduction, ligation, and extraction. The processed nucleic acid undergoes library construction and high‐throughput sequencing. Finally, the interaction information between chromatin fragments can be revealed through the sequencing data analysis. c‐MoKa allows genomic sequences to be mapped onto chromosomes using only a single individual, facilitating chromosomal‐level research.

In this study, we report a pregnant woman with balanced CCRs by c‐MoKa, Her karyotype was 46,XX,t(10;18;19)(q26.3;p11.21;p13.3). Additionally, c‐MoKa detected a unbalanced CCRs in her daughter, with a karyotype of 46,XX,der(18)t(10;18;19)(q26.3;p11.21;p13.3)dmat. This is the first case report to identify CCRs using c‐MoKa (Figure 1).

FIGURE 1.

Case identification and testing pathway: (a) Non‐Invasive Prenatal Testing (NIPT) indicates a high‐risk pregnancy. (b) Unexpected ancillary signals were detected by FISH. (c) The signal origin was determined using the c‐MoKa assay.

2. Case History/Examination

A pregnant woman visited the Department of Medical Genetics and Prenatal Diagnosis of Hainan Women and Children's Medical Center for high‐risk screening of fetal sex chromosomes via non‐invasive prenatal testing. In 2020, this woman conceived a girl naturally, who presented with ptosis, short stature, and delayed speech development after birth. The girl underwent resection of the levator palpebrae superioris, and the height was about 81 cm at age 3.5 years. However, this girl had not undergone karyotyping or any genetic tests. In 2019, the woman had a pregnancy complicated by anencephaly, which resulted in an induced abortion. The present study was approved by the Ethics Committee of Hainan Women and Children's Medical Center, and the patient provided informed consent.

3. Differential Diagnosis

Conventional karyotyping (G‐banded karyotyping) was routinely performed in our center. The peripheral blood samples of this woman and her daughter were collected for cell culture. Amniotic fluid cells were obtained via amniocentesis. G‐banding and chromosome karyotyping analysis were carried out following standard cytogenetic protocols, and karyotypes were described following the International System for Human Cytogenetics (ISCN).

FISH was performed to further evaluate the results of conventional karyotyping. The procedure was conducted at metaphase in accordance with the manufacturer's instructions, using a probe 19p13.3. All procedures were carried out under dark conditions to avoid probe photo‐bleaching and performed following the manufacturer's protocols.

Chromosome conformation capture was performed as described previously, with some modifications [3]. In brief, cells were lysed and digested with DNase I (Thermo Scientific, EN0521). Digested DNA ends were labeled with biotin‐11‐dATP (AAT Bioquest, 17014) and ligated with T4 DNA ligase (New England Biolabs, M0202V). Formaldehyde cross‐linking was reversed by incubation in 0.3% SDS and proteinase K (New England Biolabs, P8107S) for 1 h at 60°C, followed by AMPure beads (Beckman, A63881) purification. Subsequently, the result analysis was performed by Yikon Company.

4. Conclusion and Results

The conventional karyotyping result suggested that this pregnant woman might have chromosomal rearrangement involving chromosome 18 and 19, with a possible karyotype of 46,XX,t(18;19)(p11.2;p13.3) (Figure 2A). To further verify this finding, FISH was performed at metaphase for this subject. Due to probe availability, a 19p13.3 probe was used to detect the signal of p13.3 of chromosome 19. The result showed one signal of 19p13.3 on chromosome 19, with another signal detected at the terminal end of a chromosome in group C (Figure 2B). This led to the hypothesis that the subject might have a CCR undetectable by conventional karyotyping and FISH.

FIGURE 2.

G‐band karyotyping of this gravida showing the chromosomal rearrangements involving chromosome 18 and 19 (A). FISH with probe 19p13.3 showing that one signal was detected in chromosome 19, but another signal was detected in the terminal of chromosome of group C (B). Complex chromosomal rearrangements of chromosome 10, 18, and 19 were detected by c‐MoKa (C and D). Amniotic fluid karyotyping revealed a 47,XXY karyotype (E), and CNV analysis showed findings consistent with this result (F).

To verify our hypothesis, we performed c‐MoKa, which identified translocations involving chromosomes 10, 18, and 19 (Figure 2C). Additionally, no pathogenic or likely pathogenic copy number variations larger than 100 Kb were detected (Figure 2D). The final karyotype was designated as 46,XX,t(10;18;19)(q26.3;p11.21;p13.3).

The pregnant women underwent amniocentesis to determine whether her baby had any sex chromosomal abnormalities. Conventional karyotyping of amniotic fluid revealed a 47,XXY karyotype (Figure 2E). CNV analysis was consistent with karyotype findings (Figure 2F). Considering that the woman had a balanced CCR, this couple decided to continue the pregnancy. The fetus showed no abnormal ultrasound results during the follow‐up and was successfully delivered without complications on May 18, 2024.

Given the woman's history of anencephaly and her daughter's phenotype, we performed the conventional karyotyping and c‐MoKa for her daughter. Conventional karyotyping results indicated that her daughter had a unbalanced CCR (Figure 3A), while c‐MoKa analysis further identified a 15.32 Mb deletion at 18p11.32p11.1 and a 3.1 Mb duplication at 10q26.3 (Figure 3B,C). The final karyotype of the daughter was determined to be 46,XX,der(18)t(10;18;19)(q26.3;p11.21;p13.3)dmat.

FIGURE 3.

G‐band karyotyping of her daughter showing that the unbalanced chromosomal rearrangement involving chromosome 18 was detested (red arrow) (A). c‐MoKa analysis demonstrated a deletion of chromosome 18 and a duplication of chromosome 10 (indicated by red arrows in panels B and C) in the proband, both of which were determined to be of maternal origin.

5. Discussion

Complex chromosomal rearrangements (CCRs) are rare structural abnormalities that may be inherited or arise de novo [4]. They are associated with significant risks of mental retardation and phenotypic anomalies, resulting from mechanisms such as gene disruption, cryptic imbalances, or position effects [5]. Although more than half of CCRs may not manifest obvious clinical phenotypes, they are frequently diagnosed in the context of infertility, recurrent spontaneous abortion, or other reproductive issues [6]. The advent of molecular cytogenetic techniques has significantly improved the characterization of CCRs.

Conventional methods for detecting CCRs, such as karyotyping, chromosomal microarray analysis (CMA)/copy number variation (CNV) detection, and fluorescence in situ hybridization (FISH), exhibit limitations in detecting cryptic or balanced rearrangements. The emerging c‐MoKa assay can evaluate both copy number variants and structural variants, yet it cannot reliably identify balanced rearrangements and is typically confined to targeted genomic regions. Therefore, c‐MoKa is not recommended as a primary tool for evaluating CCRs in prenatal diagnostics, where comprehensive analysis is essential.

In contrast, optical genome mapping (OGM) has emerged as a superior solution [7]. It enables genome‐wide, high‐resolution detection of both balanced and unbalanced structural variants, including complex multi‐chromosomal rearrangements, making it as the preferred methodology for comprehensive prenatal genetic assessment. Consequently, any consideration of implementing c‐MoKa in a diagnostic setting must also address its time‐ and cost‐effectiveness relative to OGM, which provides more extensive information per assay despite a potentially higher initial investment.

In this study, we identified a pregnant woman with a balanced CCR and her daughter with an unbalanced CCR. To our knowledge, this is the first report demonstrating that c‐MoKa is an effective technique for testing both copy number variants and structural variants.

Author Contributions

Duo Zhou: conceptualization, data curation, formal analysis, investigation, resources, software, validation, visualization, writing – original draft. Wenchang Lian: software, writing – review and editing. Huoniao Wang: investigation, software. Renliang Huang: software. Jie Deng: conceptualization. Shujing Jiao: validation. Qiaomiao Zhou: project administration, supervision, writing – review and editing.

Funding

The authors received funding from Hainan Provincial Key R&D Program in 2023: ZDYF2024SHFZ088.

Ethics Statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of Hainan Women and children's medical center (protocol no. 2024‐195 dated 16 Sep 2024).

Consent

Written informed consent was obtained from the legal representative of the patient.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.