Detection and Haplotype Construction of ring Chromosome 17 for Carrier Screening in Preimplantation Genetic Testing

Detection and Haplotype Construction of ring Chromosome 17 for Carrier Screening in Preimplantation Genetic Testing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article Detection and Haplotype Construction of ring Chromosome 17 for Carrier Screening in Preimplantation Genetic Testing Zhiqiang Zhang, Mengnan Gu, Yijuan Huang, Taoli Ding, Shujing He, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4022562/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : While preimplantation genetic testing (PGT) carrier screening has proven efficacious in improving assisted reproduction outcomes for individuals with balanced translocations and chromosome inversions, the accurate identification of embryos carrying ring chromosomes remains a substantial challenge, undermining the efficacy of PGT. This study aims to address this gap by pioneering an advanced proband-independent haplotyping technology based on Linked-Read Sequencing technology designed to overcome the limitations of traditional molecular approaches. Results : Our integrated approach resulted in the identification of an 804 kb copy number variation (CNV) deficiency in the 17q25.3 region by CMA. Pacbio HiFi and Nanopore sequencing techniques refined breakpoint characterization, confirming telomere fusion events in chromosome 17, validated by FISH analysis. Linked-Read Sequencing technology played a pivotal role in precisely detecting CNV breakpoints and confirming end-to-end connections on chromosome 17 by linked reads. This critical insight supported the construction of a specific ring chromosome 17 haplotype for Preimplantation Genetic Testing (PGT) applications. Conclusions : This study represents a significant advancement in accurately identifying carriers of ring chromosomes and elucidating their impact on Preimplantation Genetic Testing (PGT). Our results underscore the efficacy of Linked-Read Sequencing as a robust and superior method for detecting and precisely localizing ring chromosome breakpoints, offering relatively longer DNA sequences compared to both PacBio and Nanopore technologies. Through the integration of exact breakpoint identification with haplotype linkage analysis, we were able to distinguish embryos carrying ring chromosomes from those with normal karyotypes effectively. This approach is particularly vital for chromosomes with telomeric region fusion, facilitating the construction of distinct haplotypes for affected and unaffected embryos. The methodological advancements achieved in this research significantly enhance the capabilities of PGT, marking a pivotal contribution to genetic diagnostics. Preimplantation genetic testing (PGT) Mosaic Ring chromosome 17 Haplotype construction Copy number variation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Ring chromosomes are an exceptionally rare chromosomal abnormality, primarily occurring sporadically [1]. The infrequency of individuals with ring chromosomes seeking genetic counseling for reproductive options highlights their rarity [2]. This discussion identifies two main categories of ring chromosomes: those associated with normal phenotypes and those linked to clinical presentations, including mild cognitive impairments, growth restrictions, but no severe malformations. One category involves the substitution of one homologous chromosome with a full-length or nearly full-length ring chromosome, while the other entails the addition of a derivative chromosome, typically a small ring structure containing pericentromeric chromatin, within the context of a normal karyotype. The genesis of ring chromosomes stems from two primary mechanisms. The initial mechanism involves breakage occurring in each arm of the chromosome, followed by the subsequent fusion of these fragmented ends. Alternatively, it may occur through the fusion of a broken chromosome end with the opposite telomere region, leading to genetic material loss [3, 4]. This genetic material loss, especially of terminal euchromatic regions, often results in noticeable phenotypic effects. The second mechanism involves the formation of ring chromosomes through the fusion of subtelomeric sequences, resulting in the loss of subtelomere repeat fragments, or via direct telomere-to-telomere fusion without associated deletions [5, 6]. Ring chromosomes with intact telomeres are often associated with milder phenotypes. However, even in the absence of gene deletion, telomere deletion can elicit detrimental epigenetic consequences [6, 7]. The size of the ring chromosome may can influence its stability, with larger rings potentially exhibiting lower stability compared to smaller counterparts. This instability may increase the likelihood of cellular death in vivo, raising the risk of growth failure [8, 9]. A nuanced understanding of the mechanisms and consequences of ring chromosome formation is imperative for delivering comprehensive genetic counseling and reproductive guidance in the infrequent instances where individuals or their representatives seek assistance in navigating the complexities associated with this unique chromosomal abnormality. The initial instance of a ring chromosome 17 was documented in 1974 [10]. The prevalence of such cases primarily linked to de novo occurrences and a frequent mosaic pattern due to postzygotic ring instability, leading to ring loss during cell division. In this study, we present a woman with primary infertility, characterized by a karyotype of 46, XX, r (17) (p13q25) [29]/46, XX [71]. The meiotic formation of gametes in this case could be impacted by sister chromatid exchange, potentially leading to cell division issues due to disruptions, breakages, entanglements or duplications of the ring structure, possibly resulting in aneuploidy or cell death in offspring cell [11]. In addition, her spouse carries a pathogenic mutation in the KMT2A gene. Consequently, we advocate for the implementation of preimplantation genetic testing (PGT) for this couple and introduce a novel technology designed specifically for screening embryos for ring chromosome carriers, aiming to ensure the birth of offspring with intact chromosomal profiles. Methods Study Participants In February 2022, a couple undergoing genetic counseling at our institution became integral to our research endeavor. The female partner, who served as the proband, exhibited a mosaic abnormality involving a ring chromosome 17, while the male partner carried the mutation of the KMT2A gene (NM_001197104.2: c.3190C > T, p.R1064*). Prior to participation, informed consent was rigorously obtained from all individuals or their legal guardians. We then meticulously collected peripheral blood samples from the proband and her family members, initiating extensive analytical investigations. This study obtained the ethical endorsement of the Ethics Committee at the Sixth Affiliated Hospital of Sun Yat-sen University (approval reference: 2022ZSLYEC-642). Karyotype Analysis G-banded chromosome analysis employing conventional cytogenetic techniques [12] was performed on lymphocytes extracted from the proband and their parents. A comprehensive examination was was conducted on a total of 100 cells to account for the presence of mosaicism of the ring chromosome in the proband's karyotype. Fluorescence In Situ Hybridization Analysis (FISH) We utilized the Vysis TelVysion 17q Spectrum Orange Probe for FISH analysis, specifically targeting the telomeric region of chromosome 17q (Vysis, Inc., Downers Grove, IL, USA). Fresh peripheral blood samples, which were not frozen, were diluted in normal saline, subjected to hypotonic treatment, and underwent three rounds of pre-fixation and fixation to prepare a cell suspension. The FISH procedure was conducted in strict accordance with the manufacturer’s instructions. For the ring chromosome carrier sample, a minimum of 100 cells were evaluated, with over 90% displaying a typical signal pattern, indicating a normal specimen. Conversely, more than 60% of cells exhibited an aberrant signal pattern, suggesting an anomalous sample. Chromosomal Microarray Analysis (CMA) The CMA procedure was carried out utilizing the Zhonghua 8 chip manufactured by Illumina Company, following the prescribed protocols provided by the manufacturer. The Zhonghua 8 chip features a comprehensive set of 900,000 probes, covering 81% of common variations and 60% of rare variations within the Chinese population. This platform is especially effective for genome-wide association investigations, allowing for the analysis of chromosomal copy number variations and extensive regions of loss of heterozygosity (> 10 MB). Clinical data analysis for CMA was conducted with reference to relevant literature[13]. Optical Genome Mapping (OGM) Procedures Ultra-high molecular weight DNA was extracted following the OGM protocol, which included sequence-specific fluorescent tagging of the DNA backbone. The labeled DNA was then loaded onto Saphyr chips for analysis. Instrumental operations adhered to the methodologies outlined in the referenced literature [14]. Post-sequencing analysis commenced with the preprocessing of raw data, formatted as RawMolecules.bnx.gz. Data visualization, custom analysis, and filtering of results were conducted using Bionano Access software version 1.6.1. The workflow included De Novo Assembly for chromosomal assemblies, using the software’s default parameters. Subsequent alignment against both the GRCh37/hg19 and T2T-CHM13 reference genomes was crucial for identifying chromosomal structural variants and their breakpoints. To enhance result accuracy, we applied software filtering conditions as recommended and annotated results with an internal database of a healthy population. Long Fragment Third Generation Sequencing Detection (Nanopore) We adhered to established protocols [15] for DNA extraction, library preparation, and sequencing on the Nanopore platform. The PromethION device captured electrical signal outputs which were converted to FASTQ format using the Guppy basecalling software (version 5.0.16). To enhance data integrity, NanoFilt (version 2.8.0) was employed to filter out low quality reads (Qphred ≤ 7) and short length reads (< 1000 bp), in addition to trimming 50 bp from both ends of each read. Alignment to the GRCh37/hg19 and T2T-CHM13 reference genomes was performed using Minimap2 with the following parameters: -ax map-ont -L --MD -Y -t 20. Conversion of SAM to BAM formats was carried out with SAMtools (version 1.2). Structural variations were initially identified using Sniffles, with parameters set to -t 12 --min_support 1 --num_reads_report − 1, then refined with custom scripts for integration with karyotype diagnostic reports. PEPPER-WhatsHap-DeepVariant (version r0.7-gpu) was used for detecting single nucleotide variants, InDels, and haplotyping, producing a VCF file with phasing information, with the parameter settings --ont_r9_guppy5_sup -g --phased_output -t 12. Finally, personalized python scripts were used for the classification of variants in the target region. Long Fragment Third Generation Sequencing Detection (Pacbio HiFi) The DNA extraction, library construction, and Pacbio HiFi sequencing procedures adhered to established protocols [16]. The post-sequencing data of the samples were in Fastq/bam format, and high-quality HiFi reads (QV > 20) were obtained through data filtration using SMATLink software (v11.1). The original sequence reads were aligned to the reference genome (GRCh37/hg19) using the sequence alignment software minimap2 (v2.17-r974) with default parameters to generate bam files. DeepVariant (v1.2.0) was used for analyzing single nucleotide variants (SNVs) and InDels, while pbsv software (v1.2.0) assessed structural variants with default parameters. CNV results were performed by customized CNV calling script. Haplotyping was executed using whatshap(v1.0) with default settings. This process included integrating CNV/SV breakpoint information to examine haplotype profiles of CNV variants, generating haplotypes around SNPs near the CNV region to distinguish between CNV-carrying and normal chromosomes. Proband-Independent Haplotyping Technology Based on Linked-Read Sequencing Genomic DNA extraction from peripheral blood was performed using the Nanobind CBB Big DNA Kit (Circulomics, NB-900-001-01, USA) following the manufacturer's instructions. The gDNA 165 kb Analysis Kit for the FEMTO PULS system (Agilent, FP-1002-0275, USA) assessed genomic DNA integrity and fragment size distribution. DNA library of Linked-Read Sequencing library was prepared using MGIEasystLFR Library Prep Kit (Shenzhen MGI Technology Co., Ltd.,940-000193-00, China). Qualified genomic DNA was diluted to a concentration of 1–3 ng/µl. Next, 10 ng of DNA was subjected to enzymatic fragmentation, followed by hybridization with magnetic beads tagged with molecular markers. This process fragmented the high-molecular-weight DNA molecules associated with the fragmentation enzyme. The resulting fragmented DNA was labeled with molecules, and sequencing adapters were ligated to both ends of the DNA fragments via DNA ligase, resulting in the construction of Linked-Read Sequencing libraries. Library amplification was carried out through polymerase chain reaction (PCR), and Sequencing was executed using the MGI-2000 platform (Shenzhen MGI Technology Co., Ltd., China), yielding outputs over 60 GB and a Q30 score above 90%. In the Linked-Read Sequencing data analysis workflow, the preprocessing of whole-genome sequencing data began by leveraging the unique labeling characteristics of Linked-Read Sequencing tags. This involved the removal of barcode molecular tag sequences and annotated sequences from the fastq files using a predefined barcode table. Additionally, the barcode information was encoded onto the read names within the fastq files. Low quality reads with adaptors or bases with quality of Q10 over 10% were filtered. BWA software (v0.7.17-r1188) was used to align these files against the reference genome (GRCh37/hg19), generating bam files. Post-alignment, PCR deduplication and base quality score recalibration were performed using GATK software (v4.1.4.0-local), and mutations were analyzed to produce vcf files. Prior to chromosome haplotyping, bam and vcf files were segregated for each of the 23 of chromosomes. HapCut2 software (v1.3.1) was then used in a sequential approach for assembling and haplotyping different chromosomes, integrated with results from custom CNV analysis software to identify maternal haplotypes carrying the target CNV. The mutation c.3190C > T (p.R1064*) within the KMT2A gene was identified in the parental sample sequencing data, confirming the haplotype with phased SNPs for the mutation-carrying chromosome. Given the characteristics of ring chromosomes, it is possible for sequenced long DNA fragments to span across the telomere regions from p-arm end to the other. From the reference genome alignment results, reads from extended DNA fragments with the same molecular tags showed unique alignment near both p and q arm telomere regions. By utilizing Linked-Read Sequencing's capabilities, we identified long DNA fragments with molecular tags demonstrating these characteristics, enabling the quantification of ring chromosome long DNA molecules. Linking SNP data from the ring chromosome DNA fragment sequences with phased SNPs near both telomere regions, we confirmed the SNP cluster specific to the ring chromosome and shared the same phased SNPs of the CNV-carrying haplotype. Preimplantation Genetic Testing (PGT) After removing mature oocytes, a single sperm was injected using intracytoplasmic sperm injection (ICSI) technology. The blastocysts were cultured for 5–7 days using a blastocyst medium (Vitrolife, Sweden), following protocols for blastocyst biopsy [17]. Cells (n = 6–10) were washed three times in PBS and placed in a PCR tube containing 3 µl of PBS solution, then sent to the genetics laboratory for further analysis. An integrated haplotyping-based approach for preimplantation genetic testing of embryos, in line with our study published in 2022 [18]. Embryo haplotype analysis steps are as follows. SNP loci were selected based on parental genotypes, focusing on heterozygous SNPs in one parent and homozygous in the other, to distinguish paternal and maternal alleles. These loci were used to create a haplotype table. Since the father carries KMT2A: c.3190C > T (p.R1064*) on chromosome 11, alleles were identified as F1 (mutation-carrying) and F0 (normal). Maternal CNV-carrying and normal alleles were marked M1 and M0, respectively. The constructed haplotype table for target variations was compared with SNP information from the embryos. For chromosome 11, if the embryo's paternal allele matched F1, it was colored green, otherwise colored white, to determine whether the embryo carried the risk allele F1 (Supplemental Table S1). For chromosome 17, if the embryo's maternal allele matched M1, it was colored blue, otherwise white, to determine whether the embryo carried the risk allele M1 (Supplemental Table S2). Results Clinical Presentation The proband, a 28-year-old female, has experienced primary infertility for three years without any reported familial medical history anomalies. On the other hand, the male partner, aged 29, had previously been diagnosed with mild to moderate oligoasthenospermia, along with mild intellectual disability. Whole exome sequencing revealed a pathogenic mutation, c.3190C > T (p.R1064*), in his KMT2A gene. This mutation was not found in his parents or sister through Sanger sequencing. Cytogenetic analysis for the proband and her parents showed the proband's karyotype as 46, XX, r(17)(p13q25) [29]/46, XX [71] (depicted in Fig. 1 a), with her parents exhibiting normal karyotypes. Her physical characteristics include a height of 143 cm and a weight of 52.4 kg. Upon clinical examination, sporadic café-au-lait spots were observed on her trunk and legs. No external or internal anomalies were detected, and there were no specific behavioral or motor developmental concerns. During interactions, she exhibited mild delays in speech development, but her whole exome sequencing results showed no abnormalities. Surace et al. [7]has previously reported similar observations. FISH Results The FISH examination involved the assessment of a total of 100 cells. Among these cells, 95 cells (95/100) exhibited 2 red fluorescence signals (Fig. 1 b). This finding suggests that the proband's chromosome 17 has formed a ring configuration due to terminal telomere fusion. CMA Results The CMA analysis identified a copy number deletion of 804 kb in the 17q25.3 region in the individual tested, detailed as follows: arr[GRCh37/hg19]17q25.3(80,255,858 − 81,060,040)x1, as shown in Fig. 1 c. This deletion spans 17 RefSeq protein-coding genes, including 4 Morbid genes: CYBC1, WDR45B, TBCD, and ZNF750. Notably, there is no dosage sensitivity score for this region in the ClinGen database. Furthermore, this deletion does not coincide with the general population's public database, indicating its absence in some population segments. It also does not align with pathogenic CNV regions listed in databases like Decipher and ClinGen. To date, no reports on this specific deletion have been documented in the literature. OGM Results The OGM analysis identified a deletion in the 17q25.3 region of chromosome 17, with the following results: ogm[GRCH37/hg19]17q25.3(80,323,612-qter)×1. However, it did not detect any sequences bridging the ends of chromosome 17, indicating no evidence of ring formation of chromosome 17 (depicted in Fig. 1 d). Nanopore results The patient's sequencing data yielded several critical metrics: an average genome-wide sequencing depth of 34.01-fold, an N50 read length of 35.947 kb, and an average read length of 9.965 kb. The analysis identified a chromosomal deletion at position 17q25.3. The deletion is detailed as follows: seq[GRCh37/hg19]17q25.3(80,235,078-qter)×1. This deletion is visualized in Fig. 2 a via the Integrated Genome Viewer (IGV), highlighting sequences near the CNV breakpoint at chr17:80,235,078 (hg19) and extending towards the telomere fusion zone on chromosome 17's p arm. Although there's variability in the telomere fusion region's length, the exact location of the telomeric fusion breakpoint remains undetermined. We got four reads with extending towards the telomere fusion zone but none of them include any phased SNPs in 17’s p arm. Pacbio HiFi Results The sequencing data unveiled key metrics: an average whole genome sequencing depth of 10.03X, an N50 read length of 15.45 kb, and an average read length of 10.75 kb. Utilizing PacBio technology was crucial for accurately identifying CNV deletion breakpoints, achieved by pinpointing at least two long reads that precisely located the structural variant breakpoint at 17q25.3, detailed as seq[GRCh37/hg19]17q25.3(80,235,078-qter)×1, depicted in Fig. 2 b. The unaligned segment corresponds to a repeat unit (AGGGTT) within the telomeric region. Leveraging the longest reads spanning the breakpoints, we successfully identified repeat fragments extending up to 12 kb in length. The absence of reads spanning the telomere fusion region suggests its extension beyond 12 kb. The inability of long fragments to cover the telomere fusion region hampers detecting signals for chromosome 17 ring formation. Similar to limitations with Nanopore technology, the establishment of CNV haplotypes relies solely on the deletion of CNV breakpoints, while the construction of haplotypes for ring chromosomes remains unfeasible. Linked-Read Sequencing Results The sequencing data provided crucial metrics: an average whole genome sequencing depth of 20.63X and an average fragment (reads sharing same barcodes) length of 46.69 kb. The CNV analysis from Linked-Read Sequencing concurs with CMA findings, identifying a deletion at 17q25.3, detailed as seq[GRCh37/hg19]17q25.3(80,235,078–81,195,210)×1. It's noted that Linked-Read Sequencing's breakpoint accuracy near the qter end is limited due to suboptimal alignment in this region. Upon visualizing the ring long-fragment DNA molecules using IGV, the information following the "#" symbol in each read's name represents the unique barcode tag associated with the Linked-Read Sequencing technology. In Fig. 3 a, for example, the aligned sequences of fragments of DNA molecular barcode labels 112_426_182, 255_1265_1481, and 1244_870_1276 can be located at the terminal region of q25.3 on chromosome 17, as well as the initial region of p13.3 on the same chromosome. The analysis illustrates that DNA molecules originate from a segment within the binding region of a ring chromosome, traversing from the area's leftmost part across the CNV breakpoint (Fig. 3 b). and extending to the start of 17p13.3 (Fig. 3 c). Further details concerning the ring long fragment DNA molecules are presented in Table 1. PGT Test Results Based on established parental haplotype results, SNP linkage analysis with embryo SNP data identified embryos 20222916-1-2_N2X8, F-1, and F-9 as carriers of the KMT2A mutation c.3190C > T (p.R1064*), with verification by Sanger sequencing shown in Fig. 4 a. We used embryo 20222916-1-2_N2X8 as a reference for the SNP typing result chart for the family (Fig. 4 b), highlighting the paternal risk variant-carrying chromosome in blue, allowing determination of the paternal mutation status in embryos. Specifically, embryos 20222916-1-2_N2X8, F-1, and F-9 are mutation carriers, while embryos 20222916-1-1_C1FY, F-2, F-4, F-5, F-6, F-7, and F-8 are classified as normal embryos. Unfortunately, the mutation status of the F-3 embryo could not be distinguished due to maternal ROH (1–22, X). Based on maternal CNV/ring chromosome haplotype results and SNP linkage analysis of embryo SNP data, combined with CNV scatter plot outcomes (Fig. 5 a), embryos F-3 and F-8 were identified as carriers of CNV/ring chromosomes. Utilizing F-8 as a reference, the SNP typing result chart for the family was created (Fig. 5 b), with maternal risk variant-carrying chromosomes marked in brown to determine the CNV/ring chromosome carrying status of the embryos. Consequently, we identified F-3 and F-8 as carriers, while embryos 20222916-1-1_C1FY, F-2, and F-5 were classified as normal embryos. Previous CNV analysis revealed that F-6 and F-9 exhibited a 45, XN pattern, while F-1, F-4, and F-7 carried mosaic CNVs in chromosome 17. Haplotype results identified that F-3 and F-8 carried CNV/ring chromosomes. The CNV and haplotype results of all embryos are detailed in Table 2. Prenatal Test Results The embryo labeled as 20222916-1-1_C1FY had a Euploid result and was free from the KMT2A gene mutation and ring chromosome 17. This embryo was successfully implanted into the proband's uterus, leading to a successful pregnancy. Prenatal diagnosis through amniotic fluid analysis confirmed the PGT findings. The fetal karyotype result is shown in Fig. 6 a, and the absence of the KMT2A: c.3190C > T (p.R1064*) mutation site in the fetus was verified through Sanger sequencing, as illustrated in Fig. 6 b. The embryo, identified as 20222916-1-1_C1FY, exhibited a normal CNV profile and was free from the KMT2A gene mutation and ring chromosome 17. This embryo was successfully implanted into the proband's uterus, leading to a successful pregnancy. Prenatal diagnosis through amniotic fluid analysis confirmed the PGT findings. The fetal karyotype is shown in Fig. 6 a, and the absence of the KMT2A: c.3190C > T (p.R1064*) mutation site in the fetus was verified through Sanger sequencing, as illustrated in Fig. 6 b. Discussion In this study, we explored carriers of mosaic ring chromosome 17. Chromosome karyotyping analysis confirmed a mosaicism proportion of 29% in the female carrier. CMA analysis revealed an 804 kb deletion at the 17q25.3 region on chromosome 17. FISH analysis employing the Vysis TelVysion 17q Spectrum Orange probe confirmed the telomeres' integrity at chromosome 17 ends. Long-read whole-genome sequencing technologies (PacBio and Nanopore) were designed to validate the breakpoints of the CNV on chromosome 17 and determine the status of the telomeric regions. For third generation sequencing technologies, Nanopore sequencing may detect ring chromosome DNA fusion sequences contains both p telomere regions and q telomere regions, but it may not be a robust method to construct a ring chromosome haplotype due to low base quality long DNA sequences and relative short read length. We utilized Linked-Read Sequencing to selectively target the 100 kb intervals at the ends of each chromosome, including their terminal extensions, and quantified occurrences of matching barcodes at both ends. Compared to other chromosomes, which exhibited instances occurring once or twice, chromosome 17 displayed an abundance of matching barcodes, with a total of 21 long DNA fragments and the longest fragment over 135kb length, indicating a significantly higher frequency. This discrepancy aids in the elimination of artefacts caused by end-to-end joining. Linked-Read Sequencing further confirmed that all instances of the ring chromosome presented with CNV deletion. Compared to other sequencing technologies, Linked-Read Sequencing may be a cost-effective method and play a vital role in construction of a ring chromosome haplotype with a relative long median length of DNA fragment and high quality phased SNPs by second generation sequencing platform. Integrating FISH results with Nanopore, PacBio, and Linked-Read Sequencing data confirmed telomeric fluorescence signals on the ring chromosome, supporting telomeric fusion at terminal regions as the formation mechanism. Through analysis of the patient’s mosaic chromosome karyotype, we identified three haplotypes for chromosome 17: a fully normal haplotype (100%), a haplotype with a CNV deletion at the end but without ring formation (40%), and a haplotype with a CNV deletion at the end leading to ring formation (60%). The detailed patterns are illustrated in Figure S1. The structural and behavioral instability of ring chromosomes necessitates careful consideration [19]. By analyzing the telomere lengths in the patient’s lymphocytes, we hypothesized that chromosome 17 may be predisposed to reaching a critical telomere length, thereby increasing the risk of ring formation [20]. During cell division, the unstable behavior of ring chromosomes leads to the continual production of aneuploid progeny with low viability and high rates of cell death [21]. The risk of parents with a 46,(r) karyotype having children with the same karyotype is slightly less than the theoretical 50%, with a more precise estimate being 40% [2]. Ring chromosomes can produce different types of gametes during meiosis based on the number of sister chromatid exchanges (SCEs) [22]. Zero SCEs result in symmetrical disjunction, forming a monomeric ring chromosome. Based on the CNV results, embryos F-6 and F-9 exhibited loss of chromosome 17, indicating zero sister chromatid exchanges (SCEs), potentially due to the incapacity of ring chromosome cells to persist in subsequent developmental stages, resulting in the complete loss of chromosome 17 in these embryos. One SCE results in the formation of a complete dicentric ring chromosome, and repositioning of kinetochores triggers dicentric ring breakage. Notably, no embryos in this study demonstrated the outcome of a single SCE. Two SCEs in the same direction create a figure-eight shaped dicentric ring chromosome. Repositioning of kinetochores leads to monomeric ring breakage, resulting in aneuploidy. The CNV scatter plot analysis revealed that embryos F-1, F-4, and F-7 displayed loss of mosaic chromosome 17, indicating this phenomenon. Furthermore, these embryos manifested CNV deletions at the terminal region of chromosome 17, affirming the presence of ring chromosomes with complex structural characteristics. These characteristics may be formed by two sister chromatid exchanges, where certain regions of the chromosome exhibit loss/duplication. The occurrence of sister chromatid exchanges is random, leading to dynamic rearrangements in partial or entire aneuploid cell subpopulations. These cells may subsequently die, resulting in chromosomal loss in the embryo, or survive as mosaics. Ring chromosomes are often associated with CNV deletions. Therefore, conventional methods can be used to screen embryos carrying CNV deletions and by extension serve to screen for carriers of ring chromosomes, facilitating PGT for assisted reproduction. However, for complete or nearly complete ring chromosomes, CNV testing alone cannot differentiate embryos c haplotypes, underscoring the limitations of traditional methods for PGT. Linked-Read Sequencing, with its high base quality and long DNA lengths, excels in such instances by identifying long DNA molecules that bridge ring chromosome ends, allowing for the identification of haplotypes carrying ring chromosomes and embryo genotyping. Other long-read sequencing technologies, such as PacBio, are limited by shorter read lengths (averaging about 15 kb), which are insufficient to span the telomere regions and detect long DNA molecules that connect the ends of ring chromosomes for genotyping. Although Nanopore sequencing detected signals of ring formation in this case, the limitations in read length and insufficient quantity of DNA fragments from the ring chromosome meant that evidence to support the downstream haplotype (at the 17p end) of the ring chromosome haplotype remained inconclusive. Thus, constructing the haplotype for ring chromosomes formed through telomere fusion without CNV loss becomes more challenging. Additionally, the lower accuracy of nanopore sequencing [23], renders it unreliable for haplotype genotyping. Linked-Read Sequencing Technology, by tagging long DNA fragments with transposases, while unable to tag and detect telomeric regions, allows for the most comprehensive detection of DNA molecules from ring chromosomes by aligning molecular tags with the ends of 17p and 17q. OGM technology facilitates the direct visualization of extremely long DNA molecules, enabling identifying clinically significant structural variations [24]. In the study by Tuomo et al., OGM technology successfully detected a mosaic ring X chromosome with a karyotype of 45,X[14]/46,X,r(X)(p11.21q21.1) [21]. By comparing the assembled genome map to the reference genome map, they identified signals of X chromosome ring formation [25]. However, in our study, OGM technology did not display signals of chromosome 17 ring formation. This may be due to the ring chromosome 17’s junction residing in the telomere region, which requires the ability to detect long-range signals across the telomere region to confirm chromosome ring formation. Currently, OGM technology lacks this capability, and the lower proportion of mosaic ring chromosomes in our case adds complexity to detection. In summary, utilizing long-read NGS technologies, this study delved into a female carrier of mosaic ring chromosome 17, successfully establishing the ring chromosome haplotype for embryo screening in PGT. To our knowledge, this is the first report on the haplotype analysis of ring chromosome carriers, leading to the successful birth of chromosomally normal offspring through PGT. Among 11 embryos detected, five showed deletions due to meiotic errors in ring chromosome 17. These deletions included partial loss and monosomy of chromosome 17, leading to an abnormality rate of 45.5%. Excluding one triploid embryo and two embryos unsuitable for analysis in carriers of ring chromosome 17 due to monosomy 17, the analysis of the remaining eight embryos showed that five were carriers of ring chromosome 17, with a carrier rate of 62.5%. Without PGT testing and screening for ring chromosome carriers, at least three implantation attempts would have been required to achieve a live birth of a completely chromosomally normal embryo. This study introduced innovative technological approaches, especially the application of Linked-Read Sequencing in the detection and haplotyping of ring chromosome carriers. Additionally, we explored the limitations of PacBio and Nanopore long-read sequencing technologies in this context. Conclusions Our study provides new insights into the screening and genotyping of mosaic ring chromosome carriers, offering valuable references for research and clinical applications in related fields. In the future, it is necessary to further explore the application of Linked-Read Sequencing in other samples with mosaic ring chromosomes and its adaptability in different scenarios. Declarations Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements We would like to thank all the investigators who participated in the present study. Funding This work was supported by the National Key Research and Development Program of China (2022YFC2703200). Conflict of Interests All authors declare no competing interests. References Kosztolányi, G., K. Méhes, and E.B. Hook, Inherited ring chromosomes: an analysis of published cases. Human Genetics, 1991. 87 : p. 320-324. Gardner, R.M., G.R. Sutherland, and L.G. Shaffer, Chromosome abnormalities and genetic counseling . 2012, USA: Oxford Monographs on Medical Genetics, p.201. Sigurdardottir, S., et al., Clinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of “complete ring” syndrome. American Journal of Medical Genetics, 1999. Sigurdardottir, S., et al., Clinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of “complete ring” syndrome. 1999. 87 (5): p. 384-390. Conlin, L.K., et al., Molecular analysis of ring chromosome 20 syndrome reveals two distinct groups of patients. 2011. 48 (1): p. 1-9. Henegariu, O., et al., PCR and FISH analysis of a ring Y chromosome. 1997. 69 (2): p. 171-176. Surace, C., et al., Telomere shortening and telomere position effect in mild ring 17 syndrome. Epigenetics Chromatin, 2014. 7 . Lacassie, Y., et al., Ring 2 chromosome: Ten‐year follow‐up report. 1999. 85 (2): p. 117-122. Kosztolányi, G.J.H.g., Does “ring syndrome” exist? An analysis of 207 case reports on patients with a ring autosome. 1987. 75 : p. 174-179. Ono, K., et al., A case of ring chromosome E 17: 46,XX, r(17)(p13-q25). The Japanese Journal of Human Genetics, 1975. 19 (3): p. 235-242. Knijnenburg, J., et al., Ring chromosome formation as a novel escape mechanism in patients with inverted duplication and terminal deletion. European Journal of Human Genetics Ejhg, 2007. 15 (5): p. 548-55. Lawce, H.J. and M.G.J.T.A.C.L.M. Brown, Peripheral blood cytogenetic methods. 2017: p. 87-117. Riggs, E., et al., Towards an evidence‐based process for the clinical interpretation of copy number variation. 2012. 81 (5): p. 403-412. Dremsek, P., et al., Optical genome mapping in routine human genetic diagnostics—Its advantages and limitations. 2021. 12 (12): p. 1958. Hu, L., et al., Location of balanced chromosome-translocation breakpoints by long-read sequencing on the Oxford Nanopore platform. 2020. 10 : p. 1313. Steiert, T.A., et al., High-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing. 2022. 4 (3): p. lqac051. Veiga, A., et al., Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote, 1997. 5 (4): p. 351-354. Xie, P., et al., A novel multifunctional haplotyping-based preimplantation genetic testing for different genetic conditions. 2022. 37 (11): p. 2546-2559. Kistenmacher, M.L. and H.H.J.A.J.o.H.G. Punnett, Comparative behavior of ring chromosomes. 1970. 22 (3): p. 304. Surace, C., et al., Telomere shortening and telomere position effect in mild ring 17 syndrome. 2014. 7 : p. 1-9. Bershteyn, M., et al., Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells. 2014. 507 (7490): p. 99-103. Yip, M.-Y.J.T.p., Autosomal ring chromosomes in human genetic disorders. 2015. 4 (2): p. 164. Delahaye, C. and J.J.P.o. Nicolas, Sequencing DNA with nanopores: Troubles and biases. 2021. 16 (10): p. e0257521. Sahajpal, N.S., et al., Optical genome mapping as a next-generation cytogenomic tool for detection of structural and copy number variations for prenatal genomic analyses. 2021. 12 (3): p. 398. Mantere, T., et al., Optical genome mapping enables constitutional chromosomal aberration detection. 2021. 108 (8): p. 1409-1422. Tables Tables 1 to 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files FigureS1.docx Table1.xls Table2.xls TableS1.xlsx TableS2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4022562","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":277710876,"identity":"219c4cec-cd21-495a-ab76-6fb7b3d826a8","order_by":0,"name":"Zhiqiang Zhang","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Zhiqiang","middleName":"","lastName":"Zhang","suffix":""},{"id":277710878,"identity":"1b38bc96-eb6c-421f-81fd-63c9d555a627","order_by":1,"name":"Mengnan Gu","email":"","orcid":"","institution":"Basecare Medical Device Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Mengnan","middleName":"","lastName":"Gu","suffix":""},{"id":277710879,"identity":"9f1dec2f-8437-4ec0-897b-9007ba285348","order_by":2,"name":"Yijuan Huang","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yijuan","middleName":"","lastName":"Huang","suffix":""},{"id":277710880,"identity":"c137c18b-bcd4-4ec4-a8bb-80803c99cb5e","order_by":3,"name":"Taoli Ding","email":"","orcid":"","institution":"Yikon Genomics Company, Ltd","correspondingAuthor":false,"prefix":"","firstName":"Taoli","middleName":"","lastName":"Ding","suffix":""},{"id":277710881,"identity":"2f151911-9b0d-4079-8902-8a6e960b5945","order_by":4,"name":"Shujing He","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Shujing","middleName":"","lastName":"He","suffix":""},{"id":277710882,"identity":"ae3d6ef8-ba7f-45ce-affc-133125736f1f","order_by":5,"name":"Linan Xu","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Linan","middleName":"","lastName":"Xu","suffix":""},{"id":277710883,"identity":"0b8e17ee-c0ef-48c6-bd49-5eab2053240c","order_by":6,"name":"Xiaolan Li","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolan","middleName":"","lastName":"Li","suffix":""},{"id":277710884,"identity":"d0d91020-c2ad-41d0-971b-863bb9aca06d","order_by":7,"name":"Ruixia Xu","email":"","orcid":"","institution":"Basecare Medical Device Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Ruixia","middleName":"","lastName":"Xu","suffix":""},{"id":277710885,"identity":"b69348bd-b49f-46a4-82f3-feea3053b6a1","order_by":8,"name":"Lingyin Kong","email":"","orcid":"","institution":"Basecare Medical Device Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Lingyin","middleName":"","lastName":"Kong","suffix":""},{"id":277710886,"identity":"a87b3078-1586-4e31-bfc8-cfda56bb3234","order_by":9,"name":"Haitao Zeng","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Zeng","suffix":""},{"id":277710888,"identity":"bbb7753b-0eea-4810-bc3c-8e5f0e69a205","order_by":10,"name":"Zi Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYLCCDxUMDGzscG4CYR2MM84AtTCTooWZsw1EEqvFvL338GfGedvk+ZgZmD/z/DnMwM+eY8DwcwduLTJnziUYF267bdjGzMAmzdt2mEGy540BY+8Z3FokJHIMkmduu80I0sLM23CYweBGjgEzYxseLfJvDA7zzrlt3wZzmD1BLRI8hs28DbcTgVoYpHnYgLZIENLCk2PMOOPY7eQ2oDLJuW3pPBJnnhUc7MWnhf2M8YcPNbdt57c3H/7w5o+1HH978sYHP/FoQQKMDUw8DAw8IOYBojSANf0gWukoGAWjYBSMJAAA9TlI/RsjXL4AAAAASUVORK5CYII=","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Zi","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2024-03-07 02:07:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4022562/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4022562/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52600138,"identity":"1544ed74-bdbb-4548-a7fa-062e8818ae86","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299694,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Karyotype of the proband. (b) FISH results for the proband. (c) Plot of CMA results for the proband. (d) OGM results graph for the proband.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/02de2858192f2f330fa62cfd.png"},{"id":52600587,"identity":"272691bf-667a-4a08-bb0a-39a44c9d8b28","added_by":"auto","created_at":"2024-03-13 13:00:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243129,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Nanopore integrated genome viewer (IGV) plot of the deletion breakpoint on chromosome 17. (b) Pacbio IGV plot of the deletion breakpoint on chromosome 17.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/5e442873c0e6a7378d7f86b2.png"},{"id":52600137,"identity":"8234b1f4-32e9-45ab-b01d-f96489e76d94","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":340410,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Read alignment in the ring chromosome's head-to-tail junction region. (b) Alignment of the ring chromosome's long fragment DNA molecules in the 17q25.3 region (the red box indicates the copy number variation breakpoint). (c) Alignment of the long ring chromosome fragment in the 17p13.3 region of DNA molecules (chr17:0 represents the starting position from reference 17 chromosome).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/4164b251ba2ff51778e1435e.png"},{"id":52600140,"identity":"92e1f081-8f0c-45a5-a2e2-83f9a614c612","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1353451,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Sanger verification chart of paternal KMT2A: c.3190C\u0026gt;T (p.R1064*)mutation. (b) PaternalKMT2A: c.3190C\u0026gt;T (p.R1064*) mutation single nucleotide polymorphism typing chart.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/3e968038cc294bab085f46dd.png"},{"id":52600147,"identity":"7d04a170-f163-4d12-92b8-a26e802ee741","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1206312,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Maternal ring Chromosome 17 single nucleotide polymorphism typing chart. (b) Scatterplot of embryonic copy number variation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/714ba91f38824f8854266e76.png"},{"id":52600590,"identity":"c9eae4d3-1e5b-46d6-ab7e-5aaffed3dff8","added_by":"auto","created_at":"2024-03-13 13:00:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":336228,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fetal amniotic fluid karyotype. (b) Sanger verification results for the fetal KMT2A gene c.3190C\u0026gt;T (p.R1064*) mutation site.\u003c/p\u003e\n\u003cp\u003eThe embryo, identified as 20222916-1-1_C1FY, exhibited a normal CNV profile and was free from the KMT2A gene mutation and ring chromosome 17. This embryo was successfully implanted into the proband's uterus, leading to a successful pregnancy. Prenatal diagnosis through amniotic fluid analysis confirmed the PGT findings. The fetal karyotype is shown in Figure 6a, and the absence of the KMT2A: c.3190C\u0026gt;T (p.R1064*) mutation site in the fetus was verified through Sanger sequencing, as illustrated in Figure 6b.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/08574c309af1f50a8f6b66a8.png"},{"id":78346521,"identity":"5d3ab352-26f7-4191-8f49-328032a81c21","added_by":"auto","created_at":"2025-03-12 09:39:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4854994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/efffc1da-913d-47b6-8961-5328b9f80d26.pdf"},{"id":52600585,"identity":"a082324d-78f3-4aaf-aba9-7097e283bef9","added_by":"auto","created_at":"2024-03-13 13:00:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":169565,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/97da03a084265e80b1770921.docx"},{"id":52600144,"identity":"99461525-492d-43cb-aaf4-5d15e7a3cabc","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23552,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xls","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/622fefb0b2d4e7e565fe01bb.xls"},{"id":52602563,"identity":"5b9b1a1d-7761-46aa-8651-4e36a45f6980","added_by":"auto","created_at":"2024-03-13 13:08:49","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23040,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xls","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/167741f09edee68867fc743a.xls"},{"id":52600145,"identity":"fab90246-0571-4e8d-b7f6-605a3c8dc3a2","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18382,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/37039c6c11d2a4013266c470.xlsx"},{"id":52600139,"identity":"40276bbf-e103-452d-88d3-7c4140955fdd","added_by":"auto","created_at":"2024-03-13 12:52:49","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":18240,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4022562/v1/26c49130a69dbbe10418de22.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detection and Haplotype Construction of ring Chromosome 17 for Carrier Screening in Preimplantation Genetic Testing","fulltext":[{"header":"Background","content":"\u003cp\u003eRing chromosomes are an exceptionally rare chromosomal abnormality, primarily occurring sporadically [1]. The infrequency of individuals with ring chromosomes seeking genetic counseling for reproductive options highlights their rarity [2]. This discussion identifies two main categories of ring chromosomes: those associated with normal phenotypes and those linked to clinical presentations, including mild cognitive impairments, growth restrictions, but no severe malformations. One category involves the substitution of one homologous chromosome with a full-length or nearly full-length ring chromosome, while the other entails the addition of a derivative chromosome, typically a small ring structure containing pericentromeric chromatin, within the context of a normal karyotype.\u003c/p\u003e \u003cp\u003eThe genesis of ring chromosomes stems from two primary mechanisms. The initial mechanism involves breakage occurring in each arm of the chromosome, followed by the subsequent fusion of these fragmented ends. Alternatively, it may occur through the fusion of a broken chromosome end with the opposite telomere region, leading to genetic material loss [3, 4].\u003c/p\u003e \u003cp\u003eThis genetic material loss, especially of terminal euchromatic regions, often results in noticeable phenotypic effects.\u003c/p\u003e \u003cp\u003eThe second mechanism involves the formation of ring chromosomes through the fusion of subtelomeric sequences, resulting in the loss of subtelomere repeat fragments, or via direct telomere-to-telomere fusion without associated deletions [5, 6]. Ring chromosomes with intact telomeres are often associated with milder phenotypes. However, even in the absence of gene deletion, telomere deletion can elicit detrimental epigenetic consequences [6, 7]. The size of the ring chromosome may can influence its stability, with larger rings potentially exhibiting lower stability compared to smaller counterparts. This instability may increase the likelihood of cellular death in vivo, raising the risk of growth failure [8, 9]. A nuanced understanding of the mechanisms and consequences of ring chromosome formation is imperative for delivering comprehensive genetic counseling and reproductive guidance in the infrequent instances where individuals or their representatives seek assistance in navigating the complexities associated with this unique chromosomal abnormality.\u003c/p\u003e \u003cp\u003eThe initial instance of a ring chromosome 17 was documented in 1974 [10]. The prevalence of such cases primarily linked to de novo occurrences and a frequent mosaic pattern due to postzygotic ring instability, leading to ring loss during cell division. In this study, we present a woman with primary infertility, characterized by a karyotype of 46, XX, r (17) (p13q25) [29]/46, XX [71]. The meiotic formation of gametes in this case could be impacted by sister chromatid exchange, potentially leading to cell division issues due to disruptions, breakages, entanglements or duplications of the ring structure, possibly resulting in aneuploidy or cell death in offspring cell [11]. In addition, her spouse carries a pathogenic mutation in the KMT2A gene. Consequently, we advocate for the implementation of preimplantation genetic testing (PGT) for this couple and introduce a novel technology designed specifically for screening embryos for ring chromosome carriers, aiming to ensure the birth of offspring with intact chromosomal profiles.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Participants\u003c/h2\u003e \u003cp\u003eIn February 2022, a couple undergoing genetic counseling at our institution became integral to our research endeavor. The female partner, who served as the proband, exhibited a mosaic abnormality involving a ring chromosome 17, while the male partner carried the mutation of the KMT2A gene (NM_001197104.2: c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.R1064*). Prior to participation, informed consent was rigorously obtained from all individuals or their legal guardians. We then meticulously collected peripheral blood samples from the proband and her family members, initiating extensive analytical investigations. This study obtained the ethical endorsement of the Ethics Committee at the Sixth Affiliated Hospital of Sun Yat-sen University (approval reference: 2022ZSLYEC-642).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKaryotype Analysis\u003c/h2\u003e \u003cp\u003eG-banded chromosome analysis employing conventional cytogenetic techniques [12] was performed on lymphocytes extracted from the proband and their parents. A comprehensive examination was was conducted on a total of 100 cells to account for the presence of mosaicism of the ring chromosome in the proband's karyotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence In Situ Hybridization Analysis (FISH)\u003c/h2\u003e \u003cp\u003eWe utilized the Vysis TelVysion 17q Spectrum Orange Probe for FISH analysis, specifically targeting the telomeric region of chromosome 17q (Vysis, Inc., Downers Grove, IL, USA). Fresh peripheral blood samples, which were not frozen, were diluted in normal saline, subjected to hypotonic treatment, and underwent three rounds of pre-fixation and fixation to prepare a cell suspension. The FISH procedure was conducted in strict accordance with the manufacturer\u0026rsquo;s instructions. For the ring chromosome carrier sample, a minimum of 100 cells were evaluated, with over 90% displaying a typical signal pattern, indicating a normal specimen. Conversely, more than 60% of cells exhibited an aberrant signal pattern, suggesting an anomalous sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChromosomal Microarray Analysis (CMA)\u003c/h2\u003e \u003cp\u003eThe CMA procedure was carried out utilizing the Zhonghua 8 chip manufactured by Illumina Company, following the prescribed protocols provided by the manufacturer. The Zhonghua 8 chip features a comprehensive set of 900,000 probes, covering 81% of common variations and 60% of rare variations within the Chinese population. This platform is especially effective for genome-wide association investigations, allowing for the analysis of chromosomal copy number variations and extensive regions of loss of heterozygosity (\u0026gt;\u0026thinsp;10 MB). Clinical data analysis for CMA was conducted with reference to relevant literature[13].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOptical Genome Mapping (OGM) Procedures\u003c/h2\u003e \u003cp\u003eUltra-high molecular weight DNA was extracted following the OGM protocol, which included sequence-specific fluorescent tagging of the DNA backbone. The labeled DNA was then loaded onto Saphyr chips for analysis. Instrumental operations adhered to the methodologies outlined in the referenced literature [14]. Post-sequencing analysis commenced with the preprocessing of raw data, formatted as RawMolecules.bnx.gz. Data visualization, custom analysis, and filtering of results were conducted using Bionano Access software version 1.6.1. The workflow included De Novo Assembly for chromosomal assemblies, using the software\u0026rsquo;s default parameters. Subsequent alignment against both the GRCh37/hg19 and T2T-CHM13 reference genomes was crucial for identifying chromosomal structural variants and their breakpoints. To enhance result accuracy, we applied software filtering conditions as recommended and annotated results with an internal database of a healthy population.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLong Fragment Third Generation Sequencing Detection (Nanopore)\u003c/h2\u003e \u003cp\u003eWe adhered to established protocols [15] for DNA extraction, library preparation, and sequencing on the Nanopore platform. The PromethION device captured electrical signal outputs which were converted to FASTQ format using the Guppy basecalling software (version 5.0.16). To enhance data integrity, NanoFilt (version 2.8.0) was employed to filter out low quality reads (Qphred\u0026thinsp;\u0026le;\u0026thinsp;7) and short length reads (\u0026lt;\u0026thinsp;1000 bp), in addition to trimming 50 bp from both ends of each read. Alignment to the GRCh37/hg19 and T2T-CHM13 reference genomes was performed using Minimap2 with the following parameters: -ax map-ont -L --MD -Y -t 20. Conversion of SAM to BAM formats was carried out with SAMtools (version 1.2). Structural variations were initially identified using Sniffles, with parameters set to -t 12 --min_support 1 --num_reads_report \u0026minus;\u0026thinsp;1, then refined with custom scripts for integration with karyotype diagnostic reports. PEPPER-WhatsHap-DeepVariant (version r0.7-gpu) was used for detecting single nucleotide variants, InDels, and haplotyping, producing a VCF file with phasing information, with the parameter settings --ont_r9_guppy5_sup -g --phased_output -t 12. Finally, personalized python scripts were used for the classification of variants in the target region.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLong Fragment Third Generation Sequencing Detection (Pacbio HiFi)\u003c/h2\u003e \u003cp\u003eThe DNA extraction, library construction, and Pacbio HiFi sequencing procedures adhered to established protocols [16]. The post-sequencing data of the samples were in Fastq/bam format, and high-quality HiFi reads (QV\u0026thinsp;\u0026gt;\u0026thinsp;20) were obtained through data filtration using SMATLink software (v11.1). The original sequence reads were aligned to the reference genome (GRCh37/hg19) using the sequence alignment software minimap2 (v2.17-r974) with default parameters to generate bam files. DeepVariant (v1.2.0) was used for analyzing single nucleotide variants (SNVs) and InDels, while pbsv software (v1.2.0) assessed structural variants with default parameters. CNV results were performed by customized CNV calling script. Haplotyping was executed using whatshap(v1.0) with default settings. This process included integrating CNV/SV breakpoint information to examine haplotype profiles of CNV variants, generating haplotypes around SNPs near the CNV region to distinguish between CNV-carrying and normal chromosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProband-Independent Haplotyping Technology Based on Linked-Read Sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA extraction from peripheral blood was performed using the Nanobind CBB Big DNA Kit (Circulomics, NB-900-001-01, USA) following the manufacturer's instructions. The gDNA 165 kb Analysis Kit for the FEMTO PULS system (Agilent, FP-1002-0275, USA) assessed genomic DNA integrity and fragment size distribution. DNA library of Linked-Read Sequencing library was prepared using MGIEasystLFR Library Prep Kit (Shenzhen MGI Technology Co., Ltd.,940-000193-00, China). Qualified genomic DNA was diluted to a concentration of 1\u0026ndash;3 ng/\u0026micro;l. Next, 10 ng of DNA was subjected to enzymatic fragmentation, followed by hybridization with magnetic beads tagged with molecular markers. This process fragmented the high-molecular-weight DNA molecules associated with the fragmentation enzyme. The resulting fragmented DNA was labeled with molecules, and sequencing adapters were ligated to both ends of the DNA fragments via DNA ligase, resulting in the construction of Linked-Read Sequencing libraries. Library amplification was carried out through polymerase chain reaction (PCR), and Sequencing was executed using the MGI-2000 platform (Shenzhen MGI Technology Co., Ltd., China), yielding outputs over 60 GB and a Q30 score above 90%.\u003c/p\u003e \u003cp\u003eIn the Linked-Read Sequencing data analysis workflow, the preprocessing of whole-genome sequencing data began by leveraging the unique labeling characteristics of Linked-Read Sequencing tags. This involved the removal of barcode molecular tag sequences and annotated sequences from the fastq files using a predefined barcode table. Additionally, the barcode information was encoded onto the read names within the fastq files. Low quality reads with adaptors or bases with quality of Q10 over 10% were filtered. BWA software (v0.7.17-r1188) was used to align these files against the reference genome (GRCh37/hg19), generating bam files. Post-alignment, PCR deduplication and base quality score recalibration were performed using GATK software (v4.1.4.0-local), and mutations were analyzed to produce vcf files. Prior to chromosome haplotyping, bam and vcf files were segregated for each of the 23 of chromosomes. HapCut2 software (v1.3.1) was then used in a sequential approach for assembling and haplotyping different chromosomes, integrated with results from custom CNV analysis software to identify maternal haplotypes carrying the target CNV. The mutation c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1064*) within the KMT2A gene was identified in the parental sample sequencing data, confirming the haplotype with phased SNPs for the mutation-carrying chromosome.\u003c/p\u003e \u003cp\u003eGiven the characteristics of ring chromosomes, it is possible for sequenced long DNA fragments to span across the telomere regions from p-arm end to the other. From the reference genome alignment results, reads from extended DNA fragments with the same molecular tags showed unique alignment near both p and q arm telomere regions. By utilizing Linked-Read Sequencing's capabilities, we identified long DNA fragments with molecular tags demonstrating these characteristics, enabling the quantification of ring chromosome long DNA molecules. Linking SNP data from the ring chromosome DNA fragment sequences with phased SNPs near both telomere regions, we confirmed the SNP cluster specific to the ring chromosome and shared the same phased SNPs of the CNV-carrying haplotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePreimplantation Genetic Testing (PGT)\u003c/h2\u003e \u003cp\u003eAfter removing mature oocytes, a single sperm was injected using intracytoplasmic sperm injection (ICSI) technology. The blastocysts were cultured for 5\u0026ndash;7 days using a blastocyst medium (Vitrolife, Sweden), following protocols for blastocyst biopsy [17]. Cells (n\u0026thinsp;=\u0026thinsp;6\u0026ndash;10) were washed three times in PBS and placed in a PCR tube containing 3 \u0026micro;l of PBS solution, then sent to the genetics laboratory for further analysis. An integrated haplotyping-based approach for preimplantation genetic testing of embryos, in line with our study published in 2022 [18].\u003c/p\u003e \u003cp\u003eEmbryo haplotype analysis steps are as follows. SNP loci were selected based on parental genotypes, focusing on heterozygous SNPs in one parent and homozygous in the other, to distinguish paternal and maternal alleles. These loci were used to create a haplotype table. Since the father carries KMT2A: c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1064*) on chromosome 11, alleles were identified as F1 (mutation-carrying) and F0 (normal). Maternal CNV-carrying and normal alleles were marked M1 and M0, respectively. The constructed haplotype table for target variations was compared with SNP information from the embryos. For chromosome 11, if the embryo's paternal allele matched F1, it was colored green, otherwise colored white, to determine whether the embryo carried the risk allele F1 (Supplemental Table S1). For chromosome 17, if the embryo's maternal allele matched M1, it was colored blue, otherwise white, to determine whether the embryo carried the risk allele M1 (Supplemental Table S2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical Presentation\u003c/h2\u003e\n \u003cp\u003eThe proband, a 28-year-old female, has experienced primary infertility for three years without any reported familial medical history anomalies. On the other hand, the male partner, aged 29, had previously been diagnosed with mild to moderate oligoasthenospermia, along with mild intellectual disability. Whole exome sequencing revealed a pathogenic mutation, c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1064*), in his KMT2A gene. This mutation was not found in his parents or sister through Sanger sequencing. Cytogenetic analysis for the proband and her parents showed the proband\u0026apos;s karyotype as 46, XX, r(17)(p13q25) [29]/46, XX [71] (depicted in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea), with her parents exhibiting normal karyotypes. Her physical characteristics include a height of 143 cm and a weight of 52.4 kg. Upon clinical examination, sporadic caf\u0026eacute;-au-lait spots were observed on her trunk and legs. No external or internal anomalies were detected, and there were no specific behavioral or motor developmental concerns. During interactions, she exhibited mild delays in speech development, but her whole exome sequencing results showed no abnormalities. Surace et al. [7]has previously reported similar observations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eFISH Results\u003c/h2\u003e\n \u003cp\u003eThe FISH examination involved the assessment of a total of 100 cells. Among these cells, 95 cells (95/100) exhibited 2 red fluorescence signals (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). This finding suggests that the proband\u0026apos;s chromosome 17 has formed a ring configuration due to terminal telomere fusion.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCMA Results\u003c/h2\u003e\n \u003cp\u003eThe CMA analysis identified a copy number deletion of 804 kb in the 17q25.3 region in the individual tested, detailed as follows: arr[GRCh37/hg19]17q25.3(80,255,858\u0026thinsp;\u0026minus;\u0026thinsp;81,060,040)x1, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec. This deletion spans 17 RefSeq protein-coding genes, including 4 Morbid genes: CYBC1, WDR45B, TBCD, and ZNF750. Notably, there is no dosage sensitivity score for this region in the ClinGen database. Furthermore, this deletion does not coincide with the general population\u0026apos;s public database, indicating its absence in some population segments. It also does not align with pathogenic CNV regions listed in databases like Decipher and ClinGen. To date, no reports on this specific deletion have been documented in the literature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eOGM Results\u003c/h2\u003e\n \u003cp\u003eThe OGM analysis identified a deletion in the 17q25.3 region of chromosome 17, with the following results: ogm[GRCH37/hg19]17q25.3(80,323,612-qter)\u0026times;1. However, it did not detect any sequences bridging the ends of chromosome 17, indicating no evidence of ring formation of chromosome 17 (depicted in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eNanopore results\u003c/h2\u003e\n \u003cp\u003eThe patient\u0026apos;s sequencing data yielded several critical metrics: an average genome-wide sequencing depth of 34.01-fold, an N50 read length of 35.947 kb, and an average read length of 9.965 kb. The analysis identified a chromosomal deletion at position 17q25.3. The deletion is detailed as follows: seq[GRCh37/hg19]17q25.3(80,235,078-qter)\u0026times;1. This deletion is visualized in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea via the Integrated Genome Viewer (IGV), highlighting sequences near the CNV breakpoint at chr17:80,235,078 (hg19) and extending towards the telomere fusion zone on chromosome 17\u0026apos;s p arm. Although there\u0026apos;s variability in the telomere fusion region\u0026apos;s length, the exact location of the telomeric fusion breakpoint remains undetermined. We got four reads with extending towards the telomere fusion zone but none of them include any phased SNPs in 17\u0026rsquo;s p arm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003ePacbio HiFi Results\u003c/h2\u003e\n \u003cp\u003eThe sequencing data unveiled key metrics: an average whole genome sequencing depth of 10.03X, an N50 read length of 15.45 kb, and an average read length of 10.75 kb. Utilizing PacBio technology was crucial for accurately identifying CNV deletion breakpoints, achieved by pinpointing at least two long reads that precisely located the structural variant breakpoint at 17q25.3, detailed as seq[GRCh37/hg19]17q25.3(80,235,078-qter)\u0026times;1, depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb. The unaligned segment corresponds to a repeat unit (AGGGTT) within the telomeric region. Leveraging the longest reads spanning the breakpoints, we successfully identified repeat fragments extending up to 12 kb in length. The absence of reads spanning the telomere fusion region suggests its extension beyond 12 kb. The inability of long fragments to cover the telomere fusion region hampers detecting signals for chromosome 17 ring formation. Similar to limitations with Nanopore technology, the establishment of CNV haplotypes relies solely on the deletion of CNV breakpoints, while the construction of haplotypes for ring chromosomes remains unfeasible.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eLinked-Read Sequencing Results\u003c/h2\u003e\n \u003cp\u003eThe sequencing data provided crucial metrics: an average whole genome sequencing depth of 20.63X and an average fragment (reads sharing same barcodes) length of 46.69 kb. The CNV analysis from Linked-Read Sequencing concurs with CMA findings, identifying a deletion at 17q25.3, detailed as seq[GRCh37/hg19]17q25.3(80,235,078\u0026ndash;81,195,210)\u0026times;1. It\u0026apos;s noted that Linked-Read Sequencing\u0026apos;s breakpoint accuracy near the qter end is limited due to suboptimal alignment in this region.\u003c/p\u003e\n \u003cp\u003eUpon visualizing the ring long-fragment DNA molecules using IGV, the information following the \u0026quot;#\u0026quot; symbol in each read\u0026apos;s name represents the unique barcode tag associated with the Linked-Read Sequencing technology. In Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, for example, the aligned sequences of fragments of DNA molecular barcode labels 112_426_182, 255_1265_1481, and 1244_870_1276 can be located at the terminal region of q25.3 on chromosome 17, as well as the initial region of p13.3 on the same chromosome. The analysis illustrates that DNA molecules originate from a segment within the binding region of a ring chromosome, traversing from the area\u0026apos;s leftmost part across the CNV breakpoint (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). and extending to the start of 17p13.3 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Further details concerning the ring long fragment DNA molecules are presented in Table 1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003ePGT Test Results\u003c/h2\u003e\n \u003cp\u003eBased on established parental haplotype results, SNP linkage analysis with embryo SNP data identified embryos 20222916-1-2_N2X8, F-1, and F-9 as carriers of the KMT2A mutation c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1064*), with verification by Sanger sequencing shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea. We used embryo 20222916-1-2_N2X8 as a reference for the SNP typing result chart for the family (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb), highlighting the paternal risk variant-carrying chromosome in blue, allowing determination of the paternal mutation status in embryos. Specifically, embryos 20222916-1-2_N2X8, F-1, and F-9 are mutation carriers, while embryos 20222916-1-1_C1FY, F-2, F-4, F-5, F-6, F-7, and F-8 are classified as normal embryos. Unfortunately, the mutation status of the F-3 embryo could not be distinguished due to maternal ROH (1\u0026ndash;22, X).\u003c/p\u003e\n \u003cp\u003eBased on maternal CNV/ring chromosome haplotype results and SNP linkage analysis of embryo SNP data, combined with CNV scatter plot outcomes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea), embryos F-3 and F-8 were identified as carriers of CNV/ring chromosomes. Utilizing F-8 as a reference, the SNP typing result chart for the family was created (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb), with maternal risk variant-carrying chromosomes marked in brown to determine the CNV/ring chromosome carrying status of the embryos. Consequently, we identified F-3 and F-8 as carriers, while embryos 20222916-1-1_C1FY, F-2, and F-5 were classified as normal embryos. Previous CNV analysis revealed that F-6 and F-9 exhibited a 45, XN pattern, while F-1, F-4, and F-7 carried mosaic CNVs in chromosome 17. Haplotype results identified that F-3 and F-8 carried CNV/ring chromosomes. The CNV and haplotype results of all embryos are detailed in Table 2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003ePrenatal Test Results\u003c/h2\u003e\n \u003cp\u003eThe embryo labeled as 20222916-1-1_C1FY had a Euploid result and was free from the KMT2A gene mutation and ring chromosome 17. This embryo was successfully implanted into the proband\u0026apos;s uterus, leading to a successful pregnancy. Prenatal diagnosis through amniotic fluid analysis confirmed the PGT findings. The fetal karyotype result is shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, and the absence of the KMT2A: c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1064*) mutation site in the fetus was verified through Sanger sequencing, as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb.\u003c/p\u003e\n \u003cp\u003eThe embryo, identified as 20222916-1-1_C1FY, exhibited a normal CNV profile and was free from the KMT2A gene mutation and ring chromosome 17. This embryo was successfully implanted into the proband\u0026apos;s uterus, leading to a successful pregnancy. Prenatal diagnosis through amniotic fluid analysis confirmed the PGT findings. The fetal karyotype is shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, and the absence of the KMT2A: c.3190C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.R1064*) mutation site in the fetus was verified through Sanger sequencing, as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we explored carriers of mosaic ring chromosome 17. Chromosome karyotyping analysis confirmed a mosaicism proportion of 29% in the female carrier. CMA analysis revealed an 804 kb deletion at the 17q25.3 region on chromosome 17. FISH analysis employing the Vysis TelVysion 17q Spectrum Orange probe confirmed the telomeres' integrity at chromosome 17 ends. Long-read whole-genome sequencing technologies (PacBio and Nanopore) were designed to validate the breakpoints of the CNV on chromosome 17 and determine the status of the telomeric regions. For third generation sequencing technologies, Nanopore sequencing may detect ring chromosome DNA fusion sequences contains both p telomere regions and q telomere regions, but it may not be a robust method to construct a ring chromosome haplotype due to low base quality long DNA sequences and relative short read length. We utilized Linked-Read Sequencing to selectively target the 100 kb intervals at the ends of each chromosome, including their terminal extensions, and quantified occurrences of matching barcodes at both ends. Compared to other chromosomes, which exhibited instances occurring once or twice, chromosome 17 displayed an abundance of matching barcodes, with a total of 21 long DNA fragments and the longest fragment over 135kb length, indicating a significantly higher frequency. This discrepancy aids in the elimination of artefacts caused by end-to-end joining. Linked-Read Sequencing further confirmed that all instances of the ring chromosome presented with CNV deletion. Compared to other sequencing technologies, Linked-Read Sequencing may be a cost-effective method and play a vital role in construction of a ring chromosome haplotype with a relative long median length of DNA fragment and high quality phased SNPs by second generation sequencing platform.\u003c/p\u003e \u003cp\u003eIntegrating FISH results with Nanopore, PacBio, and Linked-Read Sequencing data confirmed telomeric fluorescence signals on the ring chromosome, supporting telomeric fusion at terminal regions as the formation mechanism. Through analysis of the patient\u0026rsquo;s mosaic chromosome karyotype, we identified three haplotypes for chromosome 17: a fully normal haplotype (100%), a haplotype with a CNV deletion at the end but without ring formation (40%), and a haplotype with a CNV deletion at the end leading to ring formation (60%). The detailed patterns are illustrated in Figure S1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe structural and behavioral instability of ring chromosomes necessitates careful consideration [19]. By analyzing the telomere lengths in the patient\u0026rsquo;s lymphocytes, we hypothesized that chromosome 17 may be predisposed to reaching a critical telomere length, thereby increasing the risk of ring formation [20]. During cell division, the unstable behavior of ring chromosomes leads to the continual production of aneuploid progeny with low viability and high rates of cell death [21]. The risk of parents with a 46,(r) karyotype having children with the same karyotype is slightly less than the theoretical 50%, with a more precise estimate being 40% [2].\u003c/p\u003e \u003cp\u003eRing chromosomes can produce different types of gametes during meiosis based on the number of sister chromatid exchanges (SCEs) [22]. Zero SCEs result in symmetrical disjunction, forming a monomeric ring chromosome. Based on the CNV results, embryos F-6 and F-9 exhibited loss of chromosome 17, indicating zero sister chromatid exchanges (SCEs), potentially due to the incapacity of ring chromosome cells to persist in subsequent developmental stages, resulting in the complete loss of chromosome 17 in these embryos. One SCE results in the formation of a complete dicentric ring chromosome, and repositioning of kinetochores triggers dicentric ring breakage. Notably, no embryos in this study demonstrated the outcome of a single SCE. Two SCEs in the same direction create a figure-eight shaped dicentric ring chromosome. Repositioning of kinetochores leads to monomeric ring breakage, resulting in aneuploidy. The CNV scatter plot analysis revealed that embryos F-1, F-4, and F-7 displayed loss of mosaic chromosome 17, indicating this phenomenon. Furthermore, these embryos manifested CNV deletions at the terminal region of chromosome 17, affirming the presence of ring chromosomes with complex structural characteristics. These characteristics may be formed by two sister chromatid exchanges, where certain regions of the chromosome exhibit loss/duplication. The occurrence of sister chromatid exchanges is random, leading to dynamic rearrangements in partial or entire aneuploid cell subpopulations. These cells may subsequently die, resulting in chromosomal loss in the embryo, or survive as mosaics.\u003c/p\u003e \u003cp\u003eRing chromosomes are often associated with CNV deletions. Therefore, conventional methods can be used to screen embryos carrying CNV deletions and by extension serve to screen for carriers of ring chromosomes, facilitating PGT for assisted reproduction. However, for complete or nearly complete ring chromosomes, CNV testing alone cannot differentiate embryos c haplotypes, underscoring the limitations of traditional methods for PGT. Linked-Read Sequencing, with its high base quality and long DNA lengths, excels in such instances by identifying long DNA molecules that bridge ring chromosome ends, allowing for the identification of haplotypes carrying ring chromosomes and embryo genotyping. Other long-read sequencing technologies, such as PacBio, are limited by shorter read lengths (averaging about 15 kb), which are insufficient to span the telomere regions and detect long DNA molecules that connect the ends of ring chromosomes for genotyping. Although Nanopore sequencing detected signals of ring formation in this case, the limitations in read length and insufficient quantity of DNA fragments from the ring chromosome meant that evidence to support the downstream haplotype (at the 17p end) of the ring chromosome haplotype remained inconclusive. Thus, constructing the haplotype for ring chromosomes formed through telomere fusion without CNV loss becomes more challenging. Additionally, the lower accuracy of nanopore sequencing [23], renders it unreliable for haplotype genotyping. Linked-Read Sequencing Technology, by tagging long DNA fragments with transposases, while unable to tag and detect telomeric regions, allows for the most comprehensive detection of DNA molecules from ring chromosomes by aligning molecular tags with the ends of 17p and 17q. OGM technology facilitates the direct visualization of extremely long DNA molecules, enabling identifying clinically significant structural variations [24]. In the study by Tuomo et al., OGM technology successfully detected a mosaic ring X chromosome with a karyotype of 45,X[14]/46,X,r(X)(p11.21q21.1) [21]. By comparing the assembled genome map to the reference genome map, they identified signals of X chromosome ring formation [25]. However, in our study, OGM technology did not display signals of chromosome 17 ring formation. This may be due to the ring chromosome 17\u0026rsquo;s junction residing in the telomere region, which requires the ability to detect long-range signals across the telomere region to confirm chromosome ring formation. Currently, OGM technology lacks this capability, and the lower proportion of mosaic ring chromosomes in our case adds complexity to detection.\u003c/p\u003e \u003cp\u003eIn summary, utilizing long-read NGS technologies, this study delved into a female carrier of mosaic ring chromosome 17, successfully establishing the ring chromosome haplotype for embryo screening in PGT. To our knowledge, this is the first report on the haplotype analysis of ring chromosome carriers, leading to the successful birth of chromosomally normal offspring through PGT. Among 11 embryos detected, five showed deletions due to meiotic errors in ring chromosome 17. These deletions included partial loss and monosomy of chromosome 17, leading to an abnormality rate of 45.5%. Excluding one triploid embryo and two embryos unsuitable for analysis in carriers of ring chromosome 17 due to monosomy 17, the analysis of the remaining eight embryos showed that five were carriers of ring chromosome 17, with a carrier rate of 62.5%. Without PGT testing and screening for ring chromosome carriers, at least three implantation attempts would have been required to achieve a live birth of a completely chromosomally normal embryo. This study introduced innovative technological approaches, especially the application of Linked-Read Sequencing in the detection and haplotyping of ring chromosome carriers. Additionally, we explored the limitations of PacBio and Nanopore long-read sequencing technologies in this context.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study provides new insights into the screening and genotyping of mosaic ring chromosome carriers, offering valuable references for research and clinical applications in related fields. In the future, it is necessary to further explore the application of Linked-Read Sequencing in other samples with mosaic ring chromosomes and its adaptability in different scenarios.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the investigators who participated in the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFC2703200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKosztol\u0026aacute;nyi, G., K. M\u0026eacute;hes, and E.B. Hook, \u003cem\u003eInherited ring chromosomes: an analysis of published cases.\u003c/em\u003e Human Genetics, 1991. \u003cstrong\u003e87\u003c/strong\u003e: p. 320-324.\u003c/li\u003e\n \u003cli\u003eGardner, R.M., G.R. Sutherland, and L.G. Shaffer, \u003cem\u003eChromosome abnormalities and genetic counseling\u003c/em\u003e. 2012, USA: Oxford Monographs on Medical Genetics, p.201.\u003c/li\u003e\n \u003cli\u003eSigurdardottir, S., et al., \u003cem\u003eClinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of \u0026ldquo;complete ring\u0026rdquo; syndrome.\u003c/em\u003e American Journal of Medical Genetics, 1999.\u003c/li\u003e\n \u003cli\u003eSigurdardottir, S., et al., \u003cem\u003eClinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of \u0026ldquo;complete ring\u0026rdquo; syndrome.\u003c/em\u003e 1999. \u003cstrong\u003e87\u003c/strong\u003e(5): p. 384-390.\u003c/li\u003e\n \u003cli\u003eConlin, L.K., et al., \u003cem\u003eMolecular analysis of ring chromosome 20 syndrome reveals two distinct groups of patients.\u003c/em\u003e 2011. \u003cstrong\u003e48\u003c/strong\u003e(1): p. 1-9.\u003c/li\u003e\n \u003cli\u003eHenegariu, O., et al., \u003cem\u003ePCR and FISH analysis of a ring Y chromosome.\u003c/em\u003e 1997. \u003cstrong\u003e69\u003c/strong\u003e(2): p. 171-176.\u003c/li\u003e\n \u003cli\u003eSurace, C., et al., \u003cem\u003eTelomere shortening and telomere position effect in mild ring 17 syndrome.\u003c/em\u003e Epigenetics Chromatin, 2014. \u003cstrong\u003e7\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eLacassie, Y., et al., \u003cem\u003eRing 2 chromosome: Ten‐year follow‐up report.\u003c/em\u003e 1999. \u003cstrong\u003e85\u003c/strong\u003e(2): p. 117-122.\u003c/li\u003e\n \u003cli\u003eKosztol\u0026aacute;nyi, G.J.H.g., \u003cem\u003eDoes \u0026ldquo;ring syndrome\u0026rdquo; exist? An analysis of 207 case reports on patients with a ring autosome.\u003c/em\u003e 1987. \u003cstrong\u003e75\u003c/strong\u003e: p. 174-179.\u003c/li\u003e\n \u003cli\u003eOno, K., et al., \u003cem\u003eA case of ring chromosome E 17: 46,XX, r(17)(p13-q25).\u003c/em\u003e The Japanese Journal of Human Genetics, 1975. \u003cstrong\u003e19\u003c/strong\u003e(3): p. 235-242.\u003c/li\u003e\n \u003cli\u003eKnijnenburg, J., et al., \u003cem\u003eRing chromosome formation as a novel escape mechanism in patients with inverted duplication and terminal deletion.\u003c/em\u003e European Journal of Human Genetics Ejhg, 2007. \u003cstrong\u003e15\u003c/strong\u003e(5): p. 548-55.\u003c/li\u003e\n \u003cli\u003eLawce, H.J. and M.G.J.T.A.C.L.M. Brown, \u003cem\u003ePeripheral blood cytogenetic methods.\u003c/em\u003e 2017: p. 87-117.\u003c/li\u003e\n \u003cli\u003eRiggs, E., et al., \u003cem\u003eTowards an evidence‐based process for the clinical interpretation of copy number variation.\u003c/em\u003e 2012. \u003cstrong\u003e81\u003c/strong\u003e(5): p. 403-412.\u003c/li\u003e\n \u003cli\u003eDremsek, P., et al., \u003cem\u003eOptical genome mapping in routine human genetic diagnostics\u0026mdash;Its advantages and limitations.\u003c/em\u003e 2021. \u003cstrong\u003e12\u003c/strong\u003e(12): p. 1958.\u003c/li\u003e\n \u003cli\u003eHu, L., et al., \u003cem\u003eLocation of balanced chromosome-translocation breakpoints by long-read sequencing on the Oxford Nanopore platform.\u003c/em\u003e 2020. \u003cstrong\u003e10\u003c/strong\u003e: p. 1313.\u003c/li\u003e\n \u003cli\u003eSteiert, T.A., et al., \u003cem\u003eHigh-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing.\u003c/em\u003e 2022. \u003cstrong\u003e4\u003c/strong\u003e(3): p. lqac051.\u003c/li\u003e\n \u003cli\u003eVeiga, A., et al., \u003cem\u003eLaser blastocyst biopsy for preimplantation diagnosis in the human.\u003c/em\u003e Zygote, 1997. \u003cstrong\u003e5\u003c/strong\u003e(4): p. 351-354.\u003c/li\u003e\n \u003cli\u003eXie, P., et al., \u003cem\u003eA novel multifunctional haplotyping-based preimplantation genetic testing for different genetic conditions.\u003c/em\u003e 2022. \u003cstrong\u003e37\u003c/strong\u003e(11): p. 2546-2559.\u003c/li\u003e\n \u003cli\u003eKistenmacher, M.L. and H.H.J.A.J.o.H.G. Punnett, \u003cem\u003eComparative behavior of ring chromosomes.\u003c/em\u003e 1970. \u003cstrong\u003e22\u003c/strong\u003e(3): p. 304.\u003c/li\u003e\n \u003cli\u003eSurace, C., et al., \u003cem\u003eTelomere shortening and telomere position effect in mild ring 17 syndrome.\u003c/em\u003e 2014. \u003cstrong\u003e7\u003c/strong\u003e: p. 1-9.\u003c/li\u003e\n \u003cli\u003eBershteyn, M., et al., \u003cem\u003eCell-autonomous correction of ring chromosomes in human induced pluripotent stem cells.\u003c/em\u003e 2014. \u003cstrong\u003e507\u003c/strong\u003e(7490): p. 99-103.\u003c/li\u003e\n \u003cli\u003eYip, M.-Y.J.T.p., \u003cem\u003eAutosomal ring chromosomes in human genetic disorders.\u003c/em\u003e 2015. \u003cstrong\u003e4\u003c/strong\u003e(2): p. 164.\u003c/li\u003e\n \u003cli\u003eDelahaye, C. and J.J.P.o. Nicolas, \u003cem\u003eSequencing DNA with nanopores: Troubles and biases.\u003c/em\u003e 2021. \u003cstrong\u003e16\u003c/strong\u003e(10): p. e0257521.\u003c/li\u003e\n \u003cli\u003eSahajpal, N.S., et al., \u003cem\u003eOptical genome mapping as a next-generation cytogenomic tool for detection of structural and copy number variations for prenatal genomic analyses.\u003c/em\u003e 2021. \u003cstrong\u003e12\u003c/strong\u003e(3): p. 398.\u003c/li\u003e\n \u003cli\u003eMantere, T., et al., \u003cem\u003eOptical genome mapping enables constitutional chromosomal aberration detection.\u003c/em\u003e 2021. \u003cstrong\u003e108\u003c/strong\u003e(8): p. 1409-1422.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Preimplantation genetic testing (PGT), Mosaic, Ring chromosome 17, Haplotype construction, Copy number variation","lastPublishedDoi":"10.21203/rs.3.rs-4022562/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4022562/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: While preimplantation genetic testing (PGT) carrier screening has proven efficacious in improving assisted reproduction outcomes for individuals with balanced translocations and chromosome inversions, the accurate identification of embryos carrying ring chromosomes remains a substantial challenge, undermining the efficacy of PGT. This study aims to address this gap by pioneering an advanced proband-independent haplotyping technology based on Linked-Read Sequencing technology designed to overcome the limitations of traditional molecular approaches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Our integrated approach resulted in the identification of an 804 kb copy number variation (CNV) deficiency in the 17q25.3 region by CMA. Pacbio HiFi and Nanopore sequencing techniques refined breakpoint characterization, confirming telomere fusion events in chromosome 17, validated by FISH analysis. Linked-Read Sequencing technology played a pivotal role in precisely detecting CNV breakpoints and confirming end-to-end connections on chromosome 17 by linked reads. This critical insight supported the construction of a specific ring chromosome 17 haplotype for Preimplantation Genetic Testing (PGT) applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study represents a significant advancement in accurately identifying carriers of ring chromosomes and elucidating their impact on Preimplantation Genetic Testing (PGT). Our results underscore the efficacy of Linked-Read Sequencing as a robust and superior method for detecting and precisely localizing ring chromosome breakpoints, offering relatively longer DNA sequences compared to both PacBio and Nanopore technologies. Through the integration of exact breakpoint identification with haplotype linkage analysis, we were able to distinguish embryos carrying ring chromosomes from those with normal karyotypes effectively. This approach is particularly vital for chromosomes with telomeric region fusion, facilitating the construction of distinct haplotypes for affected and unaffected embryos. The methodological advancements achieved in this research significantly enhance the capabilities of PGT, marking a pivotal contribution to genetic diagnostics.\u003c/p\u003e","manuscriptTitle":"Detection and Haplotype Construction of ring Chromosome 17 for Carrier Screening in Preimplantation Genetic Testing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 12:52:44","doi":"10.21203/rs.3.rs-4022562/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8921765d-1cb3-4877-83b9-df078307586c","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-12T09:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-13 12:52:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4022562","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4022562","identity":"rs-4022562","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}