Introduction

Chromosomal aberrations are among the major reasons for the development of genetic diseases in humans. Owing to various factors, including physical, chemical, biological, and genetic, genomic structural variants (SVs) can occur, exhibiting changes in the gene number, ___location, or order. Furthermore, these SVs are generally related to specific phenotypes and diseases in many cases1. To date, various techniques have been employed to detect such chromosomal aberrations. Since the 1960s, karyotyping (KT) has been used to detect numerical chromosomal aberrations and SVs in clinical settings2, such as translocations, inversions, deletions, and duplications; however, its resolution is as low as 3–10 Mb3. In contrast, fluorescence in situ hybridization (FISH) presents a considerably higher resolution owing to the use of targeted probes; however, it is mainly used to validate cryptic translocations or inversions and marker chromosomes4. Notably, chromosomal microarray analysis (CMA) is a rapid and accurate pan-genome scanning technique with a high resolution; however, it presents limitations of not being able to detect balanced SVs, such as inversions, balanced translocations, and balanced rearrangements, and provide their direction and ___location5. Recently, whole-genome and long-read sequencing techniques have been employed to assess chromosomal breakpoints; however, these tests present the challenges of cost-effectiveness and data analysis.

The advantages and disadvantages of these variant-detection techniques4,5 present a potential approach of combining multiple approaches for sequential or simultaneous analysis; however, such analysis may be time-consuming and expensive. Nevertheless, approximately 60% of patients with genetic disorders remain undiagnosed6,7. Optical genome mapping (OGM) is a next-generation cytogenomic technology presenting a higher resolution (≥ 500 bp) than that of KT and CMA, and it can detect all SV types including deletions, insertions, inversions, translocations, aneuploidy, triploidy, and absence of heterozygosity8. This technique utilizes ultra-long, single-molecule DNA (≥ 150 kb) labeled at a 6-bp motif (CTTAAG), which is found every 6 kb on average throughout the genome.

This retrospective study aimed to analyze the efficacy and application potential of OGM in detecting chromosomal aberrations compared with that of conventional methods, namely KT and CMA. Herein, families who underwent preconception genetic counseling with OGM, KT, and CMA testing were included, and the results of these tests were comprehensively assessed to support etiological diagnosis or subsequent pregnancy and prevent congenital deformities.

Results

Cohort description

Herein, 37 families seeking preconception genetic counseling were enrolled, with 74 samples. The indications for genetic counseling were divided into the following four groups: pregnancy history of children with malformations or chromosomal disorders (51.4%, 19/37), recurrent miscarriages (21.6%, 8/37), or infertility (16.2%, 6/37), and family history of intellectual disability (10.8%, 4/37). KT and CMA were performed for all samples. FISH was performed in five families to validate translocations in the telomere regions or special SVs (Supplementary Table).

Quality control and variant calling of OGM

A de novo assembly pipeline was used to identify SVs and copy number variants (CNVs) in each sample. All 74 samples showed an average effective coverage (± 73.5) of 215.5× fold, average label density of 15.6/100 kb (± 0.5/100 kb), map rate of 89.5% (± 4.3%), and average N50 (> 150 Kb) of 296.3 kb (± 36.9). On average, 33 SVs and 1 CNV per sample were assessed for clinical significance after filtering (Supplementary Table).

Technical concordance between OGM and conventional methods

The results of conventional methods revealed that 16 and 21 families were positive and negative, respectively. Among the 16 positive families, 27 clinical reportable variants (15 SVs and 12 CNVs) were detected, using either one or a combination of two techniques (Table 1). OGM results presented an overall 96.3% concordance with those of conventional methods, as it missed identifying one infertility case involving a chromosome 7 and 19 centric fusion. When compared individually, OGM reached 94.7% and 100% concordance with KT and CMA, respectively.

Table 1 Concordance between optical genome mapping and conventional methods.
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Additional findings of OGM

Notably, OGM revealed additional diagnoses in three families that were negative per conventional methods and assisted in the diagnosis of six families that were positive per conventional methods. Among the three negative families, OGM revealed two cryptic balanced translocations and one small deletion, which improved the overall diagnostic rate by 5.4%. Another cryptic translocation, namely t(4;16)(p16.1;p13.3), was identified in family 14, which presented a history of pregnancies affected by chromosomal disorders (Fig. 1A), and validated by telomere FISH (Supplementary Figure A). In family 32, an additional cryptic translocation, t(8;14)(q24.3;q32.33) was detected; this family presented a history of intellectual disability (Fig. 1B). This translocation disrupted AOG2, resulting in the translocation of the first 10 exons to chromosome 14. Additionally, an 8-kb deletion that may affect SHH was detected in family 33, which presented a history of children with holoprosencephaly (HPE) (Fig. 1C). This deletion was further verified by quantitative polymerase chain reaction (qPCR) (Supplementary Figure B) and categorized as a likely pathogenic variant, being associated with the phenotype of children with malformity.

Fig. 1
figure 1

(A) Family 14. Left: Karyogram revealing a normal karyotype of 46,XX. Right: Circos plot illustrating the translocation of chromosome 4 and 16. (B) Family 32. Left: Optical genome mapping results and the genome-wide Circos plot; the pink line connecting chromosomes 8 and 14 represents the translocation. Right: Translocation of chromosome 8 and 14 disrupts AGO2 on chromosome 8. (C) Family 33. Left: Circos plot illustrating an 8-kb deletion as an orange pot (red box). Right: Genome map revealing the effects of the deletion on exons 2–3 of SHH.

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In two families that were positive per conventional methods, OGM revealed two complex rearrangements. In a family presenting a history of intellectual disability, KT detected an abnormal chromosome 6 (6p-) in the affected child. However, OGM identified an unbalanced complex rearrangement of chromosome 6 in the affected child, whereas that in the father and grandfather was found to be a balanced complex rearrangement, including inversions and insertions involving eight breakpoints. In a family presenting a history of infertility, KT detected an isochromosome of chromosome X, whereas OGM detected an additional fusion of duplicate fragment and deletion sites, correcting the karyotype result to der(X)dup(X)(p22.3p11.2)del(q26q28) (Supplementary Fig. C). Furthermore, OGM verified the presence of four cryptic translocations initially suspected by KT in four families. Overall, the rate of assisted diagnosis of OGM was 37.5% in families that tested positive for conventional methods, compared with 16.2% in all families.

Diagnostic rate

Overall, the combination of OGM and KT yielded the highest diagnostic rate of 51.4%. OGM alone achieved a diagnostic rate of 48.6%, which is higher than that of KT (37.8%) or CMA (18.9%) alone. Moreover, OGM demonstrated enhanced performance in detecting aberrations in families with a history of pregnancies involving children with malformations or chromosomal disorders, along with in families with a history of intellectual disability (Table 2).

Table 2 The diagnostic rate of different testing strategies.
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Discussion

Herein, the clinical utility of OGM was investigated in families who underwent genetic consulting for infertility, recurrent miscarriages, abnormal pregnancy history with birth defects, and family history of intellectual disability. In total, 16 families were diagnosed to be positive by conventional methods. Although OGM missed one diagnosis compared to conventional methods, it diagnosed three additional aberrations in three families, improving the overall diagnostic rate by 5.4%. Furthermore, OGM could identify the abnormalities in a single test, exhibiting a high consistency with the results of CMA combined with KT (96.3%). Among all families, OGM showed an advantage in diagnosing families with cryptic translocations, small CNVs, and complex rearrangements.

Recently, OGM has been employed in examining genetic diseases, hematological tumors, and solid tumors8,9,10,11. Additionally, various postnatal and prenatal cohort studies have proved the concordance of OGM with the combined results of CMA and KT or FISH12,13. However, there are few studies involving different preconception genetic counseling issues. Although studies on OGM have enrolled couples with infertility or recurrent miscarriages, these studies typically have a small cohort and mainly focus on detecting cryptic balanced translocations using OGM14,15. Balanced translocations in couples with recurrent miscarriages can be detected by KT, but it cannot detect cryptic balanced translocations; these can be detected by OGM, making up for the lack of diagnostic ability of KT15. Couples with a history of poor pregnancy or a family history of intellectual disability who are unable to definitively diagnose their abnormalities present various challenges in genetic counseling. Hence, in the present study, families with a pregnancy history with birth defects and a family history of intellectual disability were also included in addition to families with infertility and recurrent miscarriage. The clinical indications included in the study represent the common types encountered by genetic counseling doctors in actual preconception genetic counseling. Additionally, rather than focusing on specific types of aberration, both CNVs and SVs were comprehensively evaluated in this study.

Herein, the concordance rate of OGM with the conventional methods was 96.3% (Table 1), which is consistent with previously reported rates8,12. Furthermore, OGM showed a 5.4% increase in the diagnostic rate. Combining KT with OGM yielded the greatest diagnostic rate in families seeking preconception genetic counseling, and OGM exhibited the highest clinical diagnostic rate compared with that of KT or CMA alone. Moreover, OGM alone demonstrated a comparatively higher diagnostic rate than KT and CMA in families with a pregnancy history of children with malformations or chromosomal disorders and those with a family history of intellectual disability. Nevertheless, owing to the limited sample size, the findings of this study need to be supported by additional studies. To the best of our knowledge, no large cohort of prospective studies of families with fertility issues using OGM have been reported to date, warranting the need for such studies in the future to enhance the diagnostic rates of OGM in a more realistic context.

Reportedly, OGM presents advantages in detecting cryptic translocations and complex rearrangements15,16. A major problem plaguing couples is when one partner is a carrier of the translocation, as it presents a high probability of reproductive failure owing to the biased formation of unbalanced gametes, typically resulting in implantation failure or abortion of the established fetus17. In some cases, if the unbalanced translocation is compatible with fetal development, the child may be born with a chromosomal aberration-associated disease. These findings underscore the need to detect cryptic balanced translocations. Herein, OGM additionally identified two cryptic translocations missed by KT (in families 14 and 32) and confirmed four cryptic translocations suspected by KT (Supplementary Table). In family 32, OGM detected a cryptic translocation that disrupted AGO2, which may be attributed to the development of Lessel–Kreienkamp syndrome (LESKRES), a neurodevelopmental disorder. LESKRES is characterized by the global developmental delay with intellectual disability and speech and language delays apparent from infancy or early childhood18. A study reported a patient with LESKRES presenting a heterozygous 235.3-kb deletion involving AGO2 and an adjacent gene18. Herein, the translocation was detected in intron 10 of AGO2 (located in 8q24.3), indicating that exon 1–10 was translocated to chromosome 14. Loss of function is considered to be the pathogenic mechanism of AGO2, suggesting that the translocation is the cause of the patient phenotype and possible implications for future pregnancies. Reportedly, OGM diagnosed a family with balanced complex rearrangements in chromosome 6, with eight breaks and fusions, including insertions and inversions. These complex rearrangements were validated by FISH, utilizing specific probes, and long-range polymerase chain reaction (LR-PCR)19. In some families where balanced translocations or rearrangements were detected by OGM, the parents or children of the carriers were further examined by OGM to provide a basis for future reproductive decisions for their family members. Overall, these additional findings detected by OGM significantly contributed to enhancing the diagnosis and guidance for subsequent reproduction-related decisions of these families.

The results of this study showed that OGM could detect small deletions and duplications, highlighting its high resolution. An 8-kb deletion with a potential effect on SHH was detected in case 22OGM010 presenting twice the pregnancy history of children with HPE (Fig. 1C). The results were verified by qPCR, confirming the deletion of exons 2 and 3 of SHH (Supplementary Fig. B). Consequently, this deletion was categorized as a likely pathogenic variant. SHH encodes a secretory protein involved in establishing cell fates at several points during development, and heterozygous mutation in SHH can cause HPE3. In humans, the loss of one SHH allele is sufficient to cause HPE20, and both point mutations and microdeletions in SHH have been reported in fetuses with HPE21,22. The HPE-associated SHH mutations exhibit high heritability (73%), with 23% of parents with mutations presenting a microform23. Therefore, it was speculated that the patient included in this study might have a very mild phenotype, which when inherited in the child, made the child prone to HPE. Additionally, follow-up results showed that one of the affected children of the patient carried the deletion. The elucidation of the differential expressivity of SHH requires further investigations.

Unlike CMA, OGM can clarify the ___location and direction of the extra copy of duplicated segments or insertions, specifying the disrupted gene. These detections may facilitate the clinical interpretation of duplications, particularly intragenic ones, during diagnosis. Herein, the relative orientation and ___location of three duplications in three families could be identified using OGM, compared with the results of CMA. The duplications were as follows: one segmental duplication, as the translocated chromosome from the maternal parent was inherited; one Xp22.33p11.23 duplication that reversely rejoined to the Xq26.3q28 deletion (Supplementary Fig. C); and one tandem duplication in DMD. In family 35, a 370-kb duplication involving exon 48–56 of DMD was detected in a female by CMA; however, its positional and orientational information could not be obtained by CMA. Dystrophin-associated muscular dystrophies range from the severe Duchenne muscular dystrophy (DMD) to the milder Becker muscular dystrophy (BMD)24. Because both DMD and BMD are X-linked recessive diseases, the presence of the tandem duplication in the female suggests that the child of the female has a 50% chance of carrying the duplication and may affected. However, in case the duplicated segment of DMD does not occur within itself, for example, it is translocated to other chromosomes, then male carriers will not develop either DMD or BMD. Hence, obtaining the ___location information of the duplicated segments can help predict the clinical phenotype of the progeny of the affected individual, underscoring the potential of OGM, which could provide such information. A recent study revealed that OGM can detect similar positional information on duplications in DMD12. In this case of our study, OGM detected a tandem repeat involving exons 49–56 of DMD (Supplementary Fig. D). However, multiplex ligation-dependent probe amplification (MLPA) showed that the duplication affected exons 49–57. This could be attributed to the difference in the distribution of the CMA probe or OGM label in different DMD exons, which affected their accuracy in detecting duplications on both sides. Therefore, duplications involving parts of exons of DMD need to be carefully interpreted by combining label positions on either side of the breakpoints or verified using more accurate methods such as MLPA.

Similar to other technologies, OGM has some limitations. OGM is unable to detect centromere fusion, single nucleotide variants, and deletions or insertions of < 500 bp. Additionally, some deletions or insertions may require verification for determining pathogenicity, requiring the employment of techniques such as LR-PCR or qPCR. Nevertheless, the ability to detect variants of < 100 kb still is valuable and may be further exploited in future studies.

Conclusions

The findings of this study show that OGM technology exhibits a high concordance with conventional methods, offering notable advantages in detecting cryptic translocations and small CNVs to improve the overall diagnostic rate. Furthermore, OGM shows potential in diagnosing complex rearrangements, clarifying SVs suspected by KT, and determining the ___location and orientation of duplicated fragments. The combination of OGM and KT represents a robust diagnostic strategy for families with infertility or recurrent miscarriages. In contrast, OGM alone is sufficient for diagnosing families with a pregnancy history of children with malformations or chromosomal disorders, or a family history of intellectual disability. Altogether, these findings highlight the potential of the application of OGM in clinical settings as a rapid tool to screen families seeking preconception genetic counseling. However, for prenatal diagnosis cases where precise breakpoints need to be identified, OGM alone is not sufficient and we recommend the combined use of sequencing assays.

Materials and methods

Selection of individuals and collection of samples

In total, 74 individuals from 37 families who attended the Peking Union Medical Hospital were included in this study. The inclusion criteria were as follows: couples or families with infertility, and recurrent miscarriages; those with a pregnancy history of children with malformations, and chromosomal disorders; and those with a family history of intellectual disability. The recommended chromosomal investigations were performed per the clinical indications, such as KT and CMA, to detect SVs and submicroscopic deletions or duplications, respectively. For translocations involved in the telomere regions or special SVs, the results were validated with FISH utilizing specific probes. This study was conducted according to the Declaration of Helsinki principles and approved by the Ethics Committee of Peking Union Medical College Hospital (HS-2979).

Conventional diagnostic methods

KT and CMA were performed per the standard procedures of the diagnostic laboratories. Briefly, a previously described standard protocol was followed to perform G-banded KT25. Additionally, chromosomal aberrations were described based on the International System for Human Cytogenomic Nomenclature (ISCN 2020). CMA was performed using The Affymetrix CytoScan® 750 K array (Affymetrix, Santa Clara, CA, USA) per the instructions of the manufacturer. CNVs were classified based on the guidelines of the American College of Medical Genetics and Genomics (ACMG).

Sample processing for OGM

The Bionano Prep SP Frozen Human Blood DNA Isolation Protocol v2 (Bionano Genomics, San Diego, CA, USA) was used to extract ultra-high-molecular-weight (UHMW) DNA from whole blood or amniotic fluid cells using the SP Blood and Cell Culture DNA Isolation Kit (Bionano Genomics, San Diego, USA,80042). When the concentration of UHMW DNA was 36–150 ng/µL, the Direct Label and Stain kit (Bionano Genomics) was used per the protocol of the manufacturer to label the CTTAAG motif on 750 ng of DNA (green fluorescence), and the DNA backbone was counterstained. Qubit dsDNA HS Assay Kit and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to quantify the labeled DNA to achieve a concentration of 4–12 ng/µL, following which, the labeled DNA was loaded on a Saphyr chip (Bionano Genomics).

Quality control and data analyses for OGM

The Saphyr instrument (Bionano) was used to image and photograph the DNA loaded on the chip, and the image was converted into digital data to obtain the DNA sequence. The following four primary quality control parameters were established: molecule N50 (≥ 150 kb) > 230 kb, average label density (≥ 150 kb) per 100 kb of 14–17, map rate of molecules to the reference genome ≥ 70%, and effective genome coverage ≥ 120× fold.

Variant calling and filtering for OGM

Herein, Bionano Solve™ (v3.7) and OGM-specific pipelines managed by Bionano Access™ (v1.7) were used to analyze the SVs and CNVs in the genome by assembling molecules into consensus maps using the de novo pipeline. SVs were identified by comparing the sample consensus maps with the reference genome (hg19), and a copy number algorithm was used to detect CNVs. Following this, both CNVs and SVs were filtered according to the following parameters: (1) SVs ≤ 1500 bp; (2) SV frequency in the built-in control database < 1%; (3) minimum size of CNV ≥ 200 kb; and (4) SVs overlapping genes within 3 kb precision of the GRCh19 known canonical gene breakpoints. Filtered SVs were then analyzed and interpreted per the ACMG guidelines.

Verification methods

Standard procedures, including slide preparation, hybridization, post-hybridization washing, and counterstaining, were performed for FISH. For LR-PCR, the regions of breakpoints were amplified per the instructions of the manufacturers (Toyobo, Osaka, Japan). Thereafter, Sanger sequencing was performed to further validate the exact fusion sites of the target region.