Article Text
Abstract
Background Facioscapulohumeral muscular dystrophy 1 (FSHD1) is an autosomal dominant muscular disorder mainly caused by the contraction and hypomethylation of the D4Z4 repeat array in chromosome 4q35. Prenatal diagnosis of FSHD1 is challenging due to the highly repetitive and long genomic structure. In this study, a pregnant woman diagnosed with FSHD1 using optical genome mapping sought assistance for a healthy offspring.
Methods At the 17th week of gestation, she underwent amniocentesis, and genomic DNA (gDNA) was extracted from amniocytes. Whole-genome sequencing of the gDNA was performed using the nanopore MinION platform.
Results Despite a sequencing depth of only 7.3×, bioinformatic analyses revealed that the fetus inherited four D4Z4 repeat units with the permissive 4qA from the mother and the eight D4Z4 repeat units with the non-permissive 4qB from the father. To validate the results, SNP-based linkage analyses were conducted with gDNA from the proband, the proband’s father and proband’s amniocytes. Results indicated that the fetus inherited the maternal pathogenic haplotype based on 144 informative SNPs. Linkage analysis was consistent with the nanopore sequencing.
Conclusion Nanopore sequencing proves to be an accurate and direct method for genetic testing of monogenic diseases at the single-nucleotide level. This study represents the first application of nanopore sequencing in the prenatal diagnosis of FSHD1, providing a significant advantage for patients with de novo mutations.
- Nanopore Sequencing
Data availability statement
Data are available in a public, open access repository. The original contributions presented in this study can be found in online repositories, including the article and supplementary material. Further inquiries can be directed to the corresponding author.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
SNP-based linkage analyses and optical genome mapping have been used for prenatal diagnosis of facioscapulohumeral muscular dystrophy (FSHD).
WHAT THIS STUDY ADDS
At the single-nucleotide level, this study represents the first attempt at prenatal diagnosis of FSHD using nanopore sequencing.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
Nanopore sequencing can be used for other hereditary disorders with complex structural variations.
Introduction
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder with an incidence of approximately 1/20 000, making it the third most common muscular dystrophy.1 However, a study reported an incidence rate of 1/8500 in Netherlands.2 Wang’s team estimated a prevalence of 0.75 per million in China from 2001 to 2020, and it was the largest genetically confirmed FSHD1 population worldwide.3 According to the updated questionnaires from FSHD-CHINA, a patient advocacy and support organisation, more than 1200 individuals have been diagnosed with FSHD in China. 25.1% of individuals have not been tested (unpublished data from FSHD Peter Pan Patient Voice Report and Disease Guide). On 18 September 2023, China released the second catalogue of rare diseases, which included FSHD, providing a legal basis for enhancing the management, diagnosis and treatment of the condition, indicating that FSHD is now being considered more seriously. The disease leads to the muscular weakness and some extramuscular dysfunction.4 5 FSHD can be divided into two subtypes based on the characteristics of molecular mutations: FSHD1 (OMIM 158900) and FSHD2 (OMIM 158901). FSHD1 accounts for 95% of all patients with FSHD and is the most prevalent and classic form. It is caused by the shortened D4Z4 repeat units in the 4q35 subtelomeric region. In general, healthy individuals have 11–100 copies of D4Z4 repeats, whereas patients only have 1–10 copies of D4Z4 repeats. Approximately 5% of all patients suffer from hypomethylation and do not exhibit a truncated D4Z4 array.6 They often carry pathogenic variants of the structural maintenance of chromosomes hinge domain 1 gene (SMCHD1),7 DNA methyltransferase 3 beta (DNMT3B)8 or ligand-dependent nuclear receptor interacting factor 1 (LRIF1).9 Both FSHD1 and FSHD2 types are accompanied by changes in epigenetic modification.10 Each D4Z4 unit contains a copy of DUX4 gene (OMIM:606009), a transcription factor Double homeobox 4. The distal-most D4Z4 unit contains the full-length DUX4, which is expressed ectopically in patients with FSHD1 and FSHD2 due to epigenetic derepression. Finally, the toxic DUX4 protein can lead to muscle degeneration.7 11
The diagnosis of FSHD faces several challenges. First, it is necessary to exclude interference from the 10q26 subtelomeric region, as the homology of 4q35 and 10q26, from the upstream double homeobox 4 centromeric (DUX4c) gene to the telomere, is as high as 98%.12 13 Although the D4Z4 repeats on chromosome 10 (Chr10) can also be less than 10 units, it is not pathogenic. Second, high-frequency translocation often occurs between 4q35 and 10q26.14 Third, the length of each D4Z4 repeat unit, approximately 3.3 kb, is not suitable for traditional next-generation sequencing, posing technical challenges. Lastly, two alleles designated as 4qA and 4qB are located in the subtelomeric region, with only 4qA allele considered permissive.
The most common method for detecting FSHD is Southern blot, followed by pulsed-field gel electrophoresis, which is considered the clinical gold standard according to international guidelines.15 However, this method is time consuming and labour intensive and requires a high level of input DNA. Consequently, DNA obtained from prenatal amniotic fluid or villi often does not meet the testing requirements. Despite these drawbacks, Southern blot remains the most cost-effective method for FSHD diagnosis, particularly for patients with a family history. Molecular combing is another high-resolution technique that can be used for the direct visualisation of the D4Z4 repeat array and nearby regions using multiple colours. It has shown superior ability in detecting of mosaicism and rearrangements compared with Southern blot.16 17 This method requires specific DNA probes corresponding to the 4q35 and 10q26 loci. In recent years, optical genome mapping (OGM) has gained widespread use in clinical settings for FSHD.14 18 19 This technique can differentiate between the 4q35 and 10q26 regions, and the 4qA and 4qB alleles. Unlike Southern blot, OGM can accurately quantify the copy number of D4Z4 and identify low levels of postzygotic mosaicism. However, it requires large amounts of genomic DNA (gDNA) and is primarily used to detect peripheral blood and abortive tissue. Besides, an indirect method called karyomapping based on SNP linkage analysis has been developed for clinical practice in FSHD1.20 This technique analyses SNP loci and constructs haplotypes to identify whether the fetus carries the pathogenic chromosome. Similarly, non-invasive prenatal diagnosis of FSHD1 has been achieved using cell-free fetal DNA instead of chorionic villus or amniotic fluid.21 22 However, this method has limitations as the selected SNP loci are located 3–5 Mb upstream of the D4Z4 repeat array, making it unable to exclude the possibility of recombination between the 4q35 and 10q26 subtelomeric regions. With the development of third-generation sequencing, particularly with ultra-long reads, it has also been rapidly applied to FSHD research.23–25 This method offers direct and rapid diagnostic capabilities at the single-nucleotide level. However, prenatal diagnosis of FSHD1 based on the third-generation sequencing platforms, including nanopore sequencing, has not yet been reported.
In this study, a pregnant woman with a family history of FSHD1 sought assistance due to reproductive needs. Initially, we confirmed that she carried the pathogenic truncated D4Z4 repeats and the 4qA allele using OGM. Subsequently, she underwent the amniocentesis, and gDNA from amniotic fluid was extracted for whole-genome sequencing using the nanopore MinION platform. Our analysis revealed that the fetus inherited the same shortened D4Z4 array and the permissive 4qA allele. These results were further confirmed by constructing haplotypes. Unfortunately, SNP-based linkage analysis indicated that the fetus inherited the pathogenic haplotype from the mother and was diagnosed with FSHD1. This study provides a direct and accurate method for prenatal diagnosis of FSHD1.
Materials and methods
Patient information and ethics statement
A 24-year-old woman presented with a 2-year history of progressive limb weakness, particularly difficulty in lifting her arms over her head. She exhibited noticeable atrophy in the facial, shoulder and upper arm muscles. Electromyography revealed evidence of myogenic damages. Notably, her mother and sister also displayed similar symptoms, suggesting a hereditary form of FSHD. OGM technology was used to test the peripheral blood of the patient, revealing a shortened D4Z4 repeat unit with the 4qA allele. This analysis was done using the Saphyr system by Grandomics Biosciences, Beijing. The patient, currently in her second trimester, seeks to give birth to a healthy offspring.
This study was conducted at the Genetic and Prenatal Diagnosis Center, the First Affiliated Hospital of Zhengzhou University. The use of peripheral blood and amniotic fluid was reviewed and approved by the Research and Clinical Trials Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Written informed consent for research and publication was obtained from the index case.
DNA extraction
Amniocentesis was performed under ultrasonic positioning and guidance to obtain 15 mL of amniotic fluid. The fluid was centrifuged at 1500 rpm at room temperature for 10 min and then inoculated into a T25 culture flask at 37℃ with 5% CO2. Cells were harvested using an aseptic scraper, followed by centrifugation at 500×g for 5 min at 4℃. The cells were resuspended in 100 μL of 1× phosphate buffered saline. High molecular weight DNA was extracted using the Nanobind CBB kit (PacBio, 102-301-900), and suspended in 75 μL of nuclease-free H2O (NF H2O) as per the kit instructions. The concentration of DNA was quantified using a Qubit 4.0 fluorometer with a dsDNA HS assay kit (Thermo Fisher Scientific, Q32854). DNA purity was assessed using a Nanodrop 2000 (Thermo Fisher Scientific), and DNA length was quantified using the Qseq100 analysis system.
Library preparation and nanopore sequencing
According to the instructions of the ligation sequencing kit SQK-LSK110 (Oxford Nanopore Technologies), a higher level of input gDNA was set. Despite using the R9.4.1 version of the flow cell, a total of 2 μg gDNA was prepared for library construction. DNA repair and end prep were carried out using Agencourt AMPure XP magnetic beads (Beckman Coulter). At this stage, 61 μL of DNA was eluted, and 1 μL was used for quantification. Typically, 10% of the DNA was lost during the process. The repaired and end-prepped DNA was then subjected to the adapter ligation and clean-up steps to enrich for DNA fragments of 3 kb or longer, using the long fragment buffer. Following clean-up, approximately 20% of DNA was lost.
The MinION MK1B sequencer (Oxford Nanopore Technologies) was employed for whole-genome sequencing. Reads shorter than 200 bp were filtered out. The MinKNOW V.23.04.3 software was used to start and monitor the sequencing run for 72 hours, with pores scanning conducted every 1.5 hours. The raw electrical signal was recorded in fast5 format, with each directory containing 4000 fast5 entries. On completion of sequencing, the raw data were converted to DNA sequences (fastq) using super-high accuracy base-calling mode. Similar to our previous research,26 27 reads with mean_qscore_template ≥8 were considered to have passed the quality threshold. The filter parameter mapQ was set to ≥40. Bioinformatic analyses were performed using a custom Python script. The clean data were aligned to the human reference genome (GRCh38/hg38) using Minimap2.28 Targeted reads containing DUX4 gene and pLAM sequence were extracted by building the blast database using the makeblastdb (version 2.14.0+), and all reads were manually aligned to analyse the copy number of the D4Z4 repeat array using the NCBI blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Meanwhile, SNPs and restriction-recognition sites were identified to distinguish the Chr4 and Chr10.
Probe design for multiplex PCR
To construct haplotypes, 830 highly heterozygous SNPs (minor allele frequency >0.2) were selected based on the 1000 Genomes Project Phase 3. A specific multiplex PCR panel containing 402 loci was designed and synthesised by Nahai Bio, China. This panel covered the SNPs located at 2 Mb upstream of the D4Z4 repeat array. Amplicons were designed to avoid the repeats upstream of the D4Z4 array and distributed across different LD blocks as much as possible.
Library preparation and next-generation sequencing
The enrichment of the targeted region was performed according to the manufacturer’s protocol using multiplex PCR. gDNA from the proband, proband’s father and the fetus was extracted using the Nucleic Acid Extraction and Purification Kit (Nahai Bio, China). Library construction consisted of two steps. The first step aimed to enrich the targeted region using the specific primers through 15 cycles. The products were further amplified using primers with the universal adapter sequence at the 3’ end, with DNA barcode and sequence adapters added at both ends. The second step involved 10 cycles. The prepared library was mixed thoroughly and sequenced on the Ion Proton (Thermo Fisher, USA) with 300 sequencing flows. Each sample was assigned 1 Mb reads, with the proportion of sites with sequencing coverage exceeding 30× being >99%. The library construction kit was provided by Nahai Bio, China. The haplotype phasing of the proband was performed through family genotypes combined with Mendelian laws of inheritance. In this family, the proband’s mother was affected with FSHD according to the clinical phenotype. Therefore, SNP sites were selected where the proband’s father was homozygous and the proband was heterozygous to determine whether the fetus inherited the pathogenic chromosome by analysing 144 informative SNPs.
Methylation analysis
First, base calling was performed using the methylation mode. The targeted reads were extracted according to the reads name (two reads for 4qA and two reads for 4qB), and merged together (sub.fastq). Search the log information and find the corresponding fast5 file, and save them in the sub.fastq folder. Second, run the nanopolish command to build an index. Use minimap2 to align sub.fastq to T2T genome (GCF_009914755.1) and output the bam file. Nanopolish call methylation was performed to obtain the methy.tsv file. Third, D4Z4 was aligned to T2T genome using NCBI and the information related to Chr4 (NC_060928) was downloaded. We can get the physical locations of repeat units according to the reads name (fasta). Finally, a custom script was used to obtain the locations of 5mC for each D4Z4 unit.
Software and database
For nanopore sequencing, graphs were generated from the final report automatically generated by MinKNOW V.23.04.3. All reads (fastq) were converted to sequences (fasta) by seqtk (version 1.3-r106). Build the blast database for all.fasta using makeblastdb (version 2.14.0+). The extracted reads were manually aligned using the NCBI blast tool. Additionally, SNPs and enzyme recognition sites were analysed in detail using SnapGene V.6.0.2. For SNP linkage analysis, the raw sequencing data were aligned to the human reference genome (GRCh37/hg19) using TMAP software V.5.2.25. Figures were generated using GraphPad Prism V.9.
Results
OGM for the proband
In this family, genetic diagnosis was conducted for the proband using OGM. Results revealed that she carried the pathogenic genomic structure: four D4Z4 repeat units and the permissive 4qA allele (figure 1A). Conversely, the other haplotype was identified with 26 D4Z4 repeat units and a non-permissive 4qB allele (figure 1B). Consequently, she was diagnosed with FSHD1. Both the proband’s mother and sister displayed typical clinical symptoms. Unfortunately, her mother had passed away, and no other family members had been tested due to economic constraints. The family prioritised resources towards ensuring the birth of a healthy offspring.
Nanopore sequencing
To determine whether the fetus was affected by FSHD1, the woman underwent amniocentesis at the 17th week of gestation. gDNA was extracted from cultured amniotic fluid cells, and a gDNA library was constructed and sequenced using the nanopore MinION platform. The number of active pores dropped to zero after approximately 72 hours of running. A total of 22.8 Gb of passed bases were obtained, with an average sequencing depth of 7.3× (figure 2A). The generated reads numbered 5.96 Mb, with an estimated N50 of 6.85 kb (online supplemental figure 1). The longest 1% outliers were shown in online supplemental table 1. Candidate sequences containing full or partial-length DUX4 were extracted throughout the genome. The 4qB haplotype was distinguished by the absence of the pLAM sequence, whereas 4qA and 10q were identified using XapI and BlnI restriction enzyme sites, respectively (figure 2B). Additionally, a single-point mutation was observed in the Chr10 pLAM sequence (ATCAAA).29 We found that the first D4Z4 unit was evidently shortened in the D4Z4 array and only contained 1240 bp of the DUX4 gene. The middle D4Z4 unit contained exons 1 and 2 of DUX4 (1830 bp), and the last D4Z4 unit contained the full-length DUX4 (2072 bp), including exon 3 (online supplemental figure 2). Thus, a sequence that matched the D4Z4 repeat characteristics from the first to the last was considered valid data. Based on the limited sequencing depth, we obtained a total of 98 reads containing D4Z4 sequence. There were two full-length reads for each allele (4qA/4qB), and the raw data have been deposited in the OMIX website. Due to short sequencing length and low sequencing depth, we did not find the full-length reads for the 10q. Nanopore sequencing revealed that the fetus inherited the four D4Z4 repeat units with the pathogenic 4qA allele from its mother while carrying the eight D4Z4 repeat units with the 4qB allele, differing from the proband. It was speculated that the normal haplotype was inherited from the father, although the D4Z4 copy number of the father had not been tested previously. Furthermore, a simple sequence length polymorphism (SSLP) located 3.5 kb proximal to the first D4Z4 unit was identified. Sequence analysis showed that the fetus carried a CA10-AA-CA10 sequence, belonging to the 4qA161 haplotype. Additionally, a 4qB163 haplotype with a CA10-AA-CA11 was detected. Among the reported haplotypes, 4qA161 is the most prevalent in patients and is considered pathogenic.12
Supplemental material
SNP-based linkage analysis
To validate the accuracy of nanopore sequencing, SNP-based linkage analysis was conducted for the proband (II-2) in conjunction with her father (I-1) and the fetus (III-1). Haplotyping was employed to determine whether the fetus inherited the maternal pathogenic chromosome. A total of 144 informative SNPs were obtained (online supplemental table 2). As depicted in figure 3, the results unequivocally confirmed that the fetus inherited the pathogenic haplotype from its mother. SNP linkage analyses were consistent with the findings of nanopore sequencing.
Methylation analysis
Besides identifying the copy number of D4Z4 repeat array, we also calculated the methylation frequency for each D4Z4 unit. A gradually increased methylation (4–35%) from the proximal to the distal D4Z4 units for the permissive 4qA allele was observed. ~32% 5mC for the 4qB allele was estimated (online supplemental figure 3). Meanwhile, there were variations between two reads for each allele. This may be due to the errors generated by sequencing platform.
Discussion
FSHD is an autosomal dominant genetic disorder, with approximately 10–30% of de novo cases.3 30 Currently, prenatal diagnosis for FSHD in China is limited, with most institutions relying on indirect methods, such as linkage analysis, which necessitates multiple affected individuals in a family. However, nanopore sequencing offers a direct and pedigree-independent approach, targeting the pathogenic region at the single-nucleotide resolution, thereby surpassing traditional methods. Another advantage of nanopore sequencing is its ability to complement existing techniques, such as Southern blot, molecular combing and OGM by facilitating quantitative methylation analysis.25 31 Hypomethylation, alongside D4Z4 repeat array contraction, is a key genetic mechanism in FSHD pathogenesis, influencing the ectopic expression of the DUX4 gene.32 Studies reveal that methylation level is closely correlated with the clinical phenotype.10 33 The traditional method to test methylation is targeted bisulfite sequencing. The operation procedure is complicated, and the conversion efficiency and short-read sequencing also impact methylation status accuracy. Nanopore sequencing is sensitive to base modification.34 35 The methylation status of 4qA allele can be specifically evaluated by nanopore sequencing, which can reflect the severity and progression of the disease more accurately compared with the average methylation level measured by bisulfite sequencing. Unlike other diseases, the onset, severity and progression of FSHD are variable even between family members with the same shortened D4Z4 repeats,36 emphasising the significance of methylation assessment in investigating the relationships of genotype, phenotype and epigenetic modification. Methylation analysis also offers valuable insights into the future treatment of FSHD from a transcriptional regulation perspective.
Although the clinical phenotypes of FSHD1 and FSHD2 are similar, they exhibit varying pathogenic mechanisms at the molecular level. The diagnosis for FSHD2 required a longer DNA than that required for FSHD1, which is the crucial factor for long-read sequencing. N50 can easily reach 20–30 kb using ultra-long DNA extraction kits of Circulomics or QIAGEN.26 27 The longer the DNA fragment, the lower the sequencing depth. DNA length is negatively correlated with the sequencing depth. Nanopore sequencing for FSHD2 is more advantageous in that methylation analysis and genetic mutation of SMCHD1 or DNMT3B can be performed simultaneously when the copy number of D4Z4 repeat units does not get shortened.
In individuals of European descent, nine distinct 4q and two 10q haplotypes have been identified,12 with multiple polymorphic markers surrounding the D4Z4 repeat array contributing to chromatin structure and FSHD pathogenicity. In our study, we analysed SSLP, D4Z4 repeat number variation and 4qA/4qB allelic variation to uncover the diversity of haplotypes within the Chinese population. While SSLP is not essential for FSHD genetic diagnosis, its inclusion allows for comprehensive data mining and the identification of diverse haplotypes. Under the current hardware conditions, the depth of whole-genome sequencing was relatively low (7.3×), and the number of effective reads obtained was limited. Nevertheless, this limitation did not compromise the analysis of D4Z4 repeat number or the differentiation of haplotypes (4qA, 4qB and 10q). According to the introduction of Oxford Nanopore Technologies, for structural variation detection, we recommend sequencing to 30× depth. For single nucleotide variation (SNV) calling, we recommend sequencing to 40×−60× depth with base calling in super-high accuracy mode, proofing against sequencing errors from the system.37 38 In the light of our practice, 20×−30× depth was enough for the clinical service of FSHD. At least one valid read for each allele (4qA/4qB) should be obtained for the FSHD diagnosis. However, we cannot exclude the mosaicism with low sequencing depth. The recombination between Chr4 and Chr10 should also be approached with increased caution. Therefore, especially for the prenatal samples, we suggest another technique for retesting and verification on the basis of adequate genetic counselling. To enhance sequencing depth, Butterfield et al and Hiramuki et al combined nanopore sequencing with Cas9 enrichment, targeting centromeric (p13E-11) and telomeric (pLAM) sequences.25 31 In contrast to their strategy, from a clinical perspective, we are introducing the P2 solo based on GridION platform. With a high-output flow cell we can obtain 50–100 Gb ultra-long reads (N50>50 kb), potentially achieving up to 200 Gb throughput if the N50 is approximately 25 kb. Now the cost of a single R9.4.1 or R10.4.1 flow cell is about $2000, and the cost of a PromethION flow cell is about $2700 in China. By adding barcodes to three samples and sequencing them concurrently, we can enhance sequencing depth and significantly reduce the cost to $900. Additionally, we have developed the adaptive sampling to enrich for 4q35 and 10q26 regions (unpublished data). This is a fast and flexible method to enrich regions of interest via ejecting the off-target strands from the pore.
Nanopore sequencing is ideal for diagnosing hereditary disorders with complex structural variations, such as high sequence similarity, and tandem repeat expansions.39 It has been successfully applied in conditions such as thalassaemia,40 fragile X syndrome41 and neuromuscular disorders.42–44 Our team apply nanopore sequencing to FSHD studies earlier in China. For bioinformatic analysis, we used a custom Python script and various tools in combination, as there is currently no systematic and mature analysis software available. A limitation of this study was the small sample size. In the initial stage, we conducted genetic diagnoses of seven cases using peripheral blood based on the nanopore GridION or MinION platform; this provided valuable experience and formed the basis for prenatal studies, demonstrating the feasibility of the method. We are now focusing on prenatal diagnosis of FSHD1. In addition to amniotic fluid, we have initiated nanopore sequencing of villi at the early gestation, enabling pregnant women to make decisions earlier. This study represents the first attempt at prenatal diagnosis of FSHD1 using nanopore sequencing. We plan to recruit more patients and expand the sample size. Leveraging sequencing, we aim to provide accurate and rapid clinical services, especially for patients without a family history.
Data availability statement
Data are available in a public, open access repository. The original contributions presented in this study can be found in online repositories, including the article and supplementary material. Further inquiries can be directed to the corresponding author.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was reviewed and approved by the Research and Clinical Trials Ethics Committee of the First Affiliated Hospital of Zhengzhou University (2019-KY-286). Written informed consent was obtained from the individual for the research and publication of any potentially identifiable images or data included in this article. Participants gave informed consent to participate in the study before taking part.
Acknowledgments
This work was completed in the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University. We thank the support of Professor Xiangdong Kong and Chaoqun Yin of JC Life Science, Beijing, for their support and guidance.
References
Supplementary materials
Supplementary Data
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Footnotes
Contributors XK designed this study. DNA extraction and nanopore sequencing were performed by YW and ZZ. Bioinformatic analyses were conducted by YW and FM. SNP linkage analysis was completed by YW and ZZ. YW wrote the manuscript, and all authors discussed the results, revised the manuscript and approved the submission. XK was the guarantor.
Funding This work was supported by the Henan Province Medical Science and Technique Foundation (SBGJ202102097), the Henan Province Fertility Protection and Eugenics Key Laboratory Open Project (SYLBHHYS2022-02), the Guangxi Key Laboratory of Birth Defects Open Project (GXWCH-ZDKF-2022-05) and the Natural Science Foundation of Henan Province (242300420400).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.