光学基因组图谱用于破译寻求孕前遗传咨询的家庭中的染色体畸变-动脉网

Optical genome mapping to decipher the chromosomal aberrations in families seeking for preconception genetic counseling

Nature 等信源发布 2025-01-21 17:23

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Abstract

摘要

Optical genome mapping (OGM) offers high consistency in simultaneously detecting structural and copy number variants. This study aimed to retrospectively evaluate the efficacy and potential applications of OGM in preconception genetic counseling. Herein, 74 samples from 37 families were included, and their results of OGM were compared to conventional methods, namely karyotyping (KT) and chromosomal microarray analysis (CMA), which identified 27 variants across 16 positive families.

。这项研究旨在回顾性评估OGM在孕前遗传咨询中的功效和潜在应用。在此，纳入了来自37个家族的74个样本，并将其OGM结果与常规方法（即核型分析（KT）和染色体微阵列分析（CMA））进行了比较，后者在16个阳性家族中鉴定出27个变体。

Notably, OGM achieved a concordance rate of 94.7% and 100% with KT and CMA, respectively, presenting an overall concordance of 96.3%, as it missed detecting a centromeric translocation. Additionally, OGM detected two cryptic balanced translocations and a small deletion in three families that were missed by conventional methods, improving the diagnostic rate by 5.4%, along with assisting in the diagnoses of six families (16.2%) by identifying complex rearrangements and confirming cryptic translocations.

值得注意的是，OGM与KT和CMA的一致率分别为94.7%和100%，总体一致率为96.3%，因为它没有检测到着丝粒易位。此外，OGM在常规方法遗漏的三个家族中检测到两个隐性平衡易位和一个小缺失，将诊断率提高了5.4%，同时通过识别复杂的重排和确认隐性易位来帮助诊断六个家族（16.2%）。

The combination of KT with OGM yielded the highest diagnostic rate in all families. Overall, the findings of this study present the notable potential of OGM for its application, combined with KT per requirement, in clinical settings to improve the efficiency and accuracy of diagnoses and rapid screening of individuals seeking preconception genetic counseling..

KT与OGM的结合在所有家庭中产生了最高的诊断率。总体而言，这项研究的结果表明，OGM在临床环境中具有显着的应用潜力，结合KT的要求，可以提高诊断的效率和准确性，并快速筛查寻求孕前遗传咨询的个体。。

Introduction

简介

Chromosomal aberrations are among the major reasons for the development of genetic diseases in humans. Owing to various factors, including physical, chemical, biological, and genetic, genomic structural variants (SVs) can occur, exhibiting changes in the gene number, location, or order. Furthermore, these SVs are generally related to specific phenotypes and diseases in many cases.

染色体畸变是人类遗传疾病发展的主要原因之一。由于各种因素，包括物理，化学，生物和遗传，可能会发生基因组结构变异（SV），表现出基因数量，位置或顺序的变化。此外，在许多情况下，这些SV通常与特定的表型和疾病有关。

. To date, various techniques have been employed to detect such chromosomal aberrations. Since the 1960s, karyotyping (KT) has been used to detect numerical chromosomal aberrations and SVs in clinical settings

迄今为止，已经采用了各种技术来检测这种染色体畸变。自20世纪60年代以来，核型分析（KT）已被用于检测临床环境中的数值染色体畸变和SV

, such as translocations, inversions, deletions, and duplications; however, its resolution is as low as 3–10 Mb

，例如易位，倒位，缺失和重复；但是，它的分辨率低至3-10 Mb

. In contrast, fluorescence in situ hybridization (FISH) presents a considerably higher resolution owing to the use of targeted probes; however, it is mainly used to validate cryptic translocations or inversions and marker chromosomes

相反，由于使用了靶向探针，荧光原位杂交（FISH）的分辨率要高得多；然而，它主要用于验证隐性易位或倒位以及标记染色体

. Notably, chromosomal microarray analysis (CMA) is a rapid and accurate pan-genome scanning technique with a high resolution; however, it presents limitations of not being able to detect balanced SVs, such as inversions, balanced translocations, and balanced rearrangements, and provide their direction and location.

；但是，它存在无法检测平衡SV（例如倒位，平衡易位和平衡重排）并提供其方向和位置的局限性。

. Recently, whole-genome and long-read sequencing techniques have been employed to assess chromosomal breakpoints; however, these tests present the challenges of cost-effectiveness and data analysis.

最近，全基因组和长读测序技术已被用于评估染色体断裂点；然而，这些测试带来了成本效益和数据分析的挑战。

The advantages and disadvantages of these variant-detection techniques

这些变异检测技术的优缺点

present a potential approach of combining multiple approaches for sequential or simultaneous analysis; however, such analysis may be time-consuming and expensive. Nevertheless, approximately 60% of patients with genetic disorders remain undiagnosed

提出了一种结合多种方法进行顺序或同时分析的潜在方法；但是，这种分析可能既费时又昂贵。然而，大约60%的遗传性疾病患者仍未确诊

. Optical genome mapping (OGM) is a next-generation cytogenomic technology presenting a higher resolution (≥ 500 bp) than that of KT and CMA, and it can detect all SV types including deletions, insertions, inversions, translocations, aneuploidy, triploidy, and absence of heterozygosity

光学基因组作图（OGM）是一种比KT和CMA具有更高分辨率（≥500 bp）的下一代细胞基因组技术，它可以检测所有SV类型，包括缺失、插入、倒位、易位、非整倍性、三倍体和无杂合性

. This technique utilizes ultra-long, single-molecule DNA (≥ 150 kb) labeled at a 6-bp motif (CTTAAG), which is found every 6 kb on average throughout the genome.

该技术利用以6 bp基序（CTTAAG）标记的超长单分子DNA（≥150 kb），在整个基因组中平均每6 kb发现一次。

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

。在此，纳入了接受OGM，KT和CMA检测的孕前遗传咨询的家庭，并对这些检测的结果进行了全面评估，以支持病因诊断或随后的妊娠并预防先天性畸形。。

Results

结果

Cohort description

队列描述

Herein, 37 families seeking preconception genetic counseling were enrolled, with 74 samples. The indications for genetic counseling were divided into the following four groups: pregnancy history of children with malformations or chromosomal disorders (51.4%, 19/37), recurrent miscarriages (21.6%, 8/37), or infertility (16.2%, 6/37), and family history of intellectual disability (10.8%, 4/37).

在此，招募了37个寻求孕前遗传咨询的家庭，共74个样本。遗传咨询的适应症分为以下四组：畸形或染色体异常儿童的妊娠史（51.4%，19/37），反复流产（21.6%，8/37）或不孕症（16.2%，6/37）和智力障碍家族史（10.8%，4/37）。

KT and CMA were performed for all samples. FISH was performed in five families to validate translocations in the telomere regions or special SVs (Supplementary Table)..

对所有样品进行KT和CMA。FISH在五个家族中进行，以验证端粒区域或特殊SV的易位（补充表）。。

Quality control and variant calling of OGM

A de novo assembly pipeline was used to identify SVs and copy number variants (CNVs) in each sample. All 74 samples showed an average effective coverage (± 73.5) of 215.5× fold, average label density of 15.6/100 kb (± 0.5/100 kb), map rate of 89.5% (± 4.3%), and average N50 (> 150 Kb) of 296.3 kb (± 36.9).

从头组装管道用于识别每个样品中的SV和拷贝数变体（CNV）。所有74个样本的平均有效覆盖率（±73.5）为215.5倍，平均标记密度为15.6/100 kb（±0.5/100 kb），map率为89.5%（±4.3%），平均N50（>150 kb）为296.3 kb（±36.9）。

On average, 33 SVs and 1 CNV per sample were assessed for clinical significance after filtering (Supplementary Table)..

过滤后，平均每个样本评估33个SV和1个CNV的临床意义（补充表）。。

Technical concordance between OGM and conventional methods

OGM与常规方法的技术一致性

The results of conventional methods revealed that 16 and 21 families were positive and negative, respectively. Among the 16 positive families, 27 clinical reportable variants (15 SVs and 12 CNVs) were detected, using either one or a combination of two techniques (Table

常规方法的结果显示，分别有16个和21个家庭呈阳性和阴性。在16个阳性家族中，使用一种或两种技术的组合检测到27个临床可报告变异（15个SV和12个CNV）（表

). OGM results presented an overall 96.3% concordance with those of conventional methods, as it missed identifying one infertility case involving a chromosome 7 and 19 centric fusion. When compared individually, OGM reached 94.7% and 100% concordance with KT and CMA, respectively.

)。OGM结果与常规方法的总体一致性为96.3%，因为它错过了一例涉及7号和19号染色体中心融合的不孕症病例。单独比较时，OGM与KT和CMA的一致性分别达到94.7%和100%。

Table 1 Concordance between optical genome mapping and conventional methods.

表1光学基因组作图与常规方法之间的一致性。

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Additional findings of OGM

OGM的其他发现

Notably, OGM revealed additional diagnoses in three families that were negative per conventional methods and assisted in the diagnosis of six families that were positive per conventional methods. Among the three negative families, OGM revealed two cryptic balanced translocations and one small deletion, which improved the overall diagnostic rate by 5.4%.

值得注意的是，OGM在三个常规方法阴性的家庭中发现了其他诊断，并协助诊断了六个常规方法阳性的家庭。在这三个阴性家族中，OGM显示出两个隐性平衡易位和一个小缺失，这使总体诊断率提高了5.4%。

Another cryptic translocation, namely t(4;16)(p16.1;p13.3), was identified in family 14, which presented a history of pregnancies affected by chromosomal disorders (Fig. .

在家族14中发现了另一种隐性易位，即t（4；16）（p16.1；p13.3），该家族表现出受染色体疾病影响的妊娠史（图。

A), and validated by telomere FISH (Supplementary Figure A). In family 32, an additional cryptic translocation, t(8;14)(q24.3;q32.33) was detected; this family presented a history of intellectual disability (Fig.

A），并通过端粒FISH验证（补充图A）。在家族32中，检测到另一个隐性易位t（8；14）（q24.3；q32.33）；这个家庭有智力残疾史（图）。

B). This translocation disrupted

B）。这种易位破坏了

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, resulting in the translocation of the first 10 exons to chromosome 14. Additionally, an 8-kb deletion that may affect

，导致前10个外显子易位至14号染色体。此外，8 kb的删除可能会影响

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was detected in family 33, which presented a history of children with holoprosencephaly (HPE) (Fig.

在家族33中检测到，该家族有全前脑（HPE）儿童的病史（图）。

C). This deletion was further verified by quantitative polymerase chain reaction (qPCR) (Supplementary Figure B) and categorized as a likely pathogenic variant, being associated with the phenotype of children with malformity.

C）。通过定量聚合酶链反应（qPCR）进一步验证了这种缺失（补充图B），并将其归类为可能的致病变异，与畸形儿童的表型有关。

Fig. 1

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(

) Family 14. Left: Karyogram revealing a normal karyotype of 46,XX. Right: Circos plot illustrating the translocation of chromosome 4 and 16. (

)家庭14。左：核型图显示正常核型为46，XX。右：Circos图说明了4号和16号染色体的易位。（笑声）(

B类

) Family 32. Left: Optical genome mapping results and the genome-wide Circos plot; the pink line connecting chromosomes 8 and 14 represents the translocation. Right: Translocation of chromosome 8 and 14 disrupts

)家庭32。左：光学基因组作图结果和全基因组Circos图；连接染色体8和14的粉红色线代表易位。右：8号和14号染色体的易位破坏了

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on chromosome 8. (

在8号染色体上。（笑声）(

C级

) Family 33. Left: Circos plot illustrating an 8-kb deletion as an orange pot (red box). Right: Genome map revealing the effects of the deletion on exons 2–3 of

)。左：Circos图，显示了一个8 kb的删除，作为一个橙色的锅（红色框）。右：基因组图谱揭示了缺失对外显子2-3的影响

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In two families that were positive per conventional methods, OGM revealed two complex rearrangements. In a family presenting a history of intellectual disability, KT detected an abnormal chromosome 6 (6p-) in the affected child. However, OGM identified an unbalanced complex rearrangement of chromosome 6 in the affected child, whereas that in the father and grandfather was found to be a balanced complex rearrangement, including inversions and insertions involving eight breakpoints.

在按常规方法呈阳性的两个家族中，OGM揭示了两个复杂的重排。在一个有智力障碍史的家庭中，KT在患病儿童中检测到异常的6号染色体（6p-）。但是，OGM在患病儿童中发现了6号染色体的不平衡复杂重排，而在父亲和祖父中则发现了平衡的复杂重排，包括涉及八个断点的倒位和插入。

In a family presenting a history of infertility, KT detected an isochromosome of chromosome X, whereas OGM detected an additional fusion of duplicate fragment and deletion sites, correcting the karyotype result to der(X)dup(X)(p22.3p11.2)del(q26q28) (Supplementary Fig. C). Furthermore, OGM verified the presence of four cryptic translocations initially suspected by KT in four families.

在一个有不孕史的家族中，KT检测到X染色体的等染色体，而OGM检测到重复片段和缺失位点的额外融合，将核型结果校正为der（X）dup（X）（p22.3p11.2）del（q26q28）（补充图C）。此外，OGM证实了KT最初在四个家族中怀疑存在四个隐性易位。

Overall, the rate of assisted diagnosis of OGM was 37.5% in families that tested positive for conventional methods, compared with 16.2% in all families..

总体而言，常规方法检测呈阳性的家庭中，OGM的辅助诊断率为37.5%，而所有家庭的辅助诊断率为16.2%。。

Diagnostic rate

诊断率

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

总体而言，OGM和KT的联合诊断率最高，为51.4%。单独使用OGM的诊断率为48.6%，高于单独使用KT（37.8%）或CMA（18.9%）。此外，OGM在检测有畸形或染色体异常儿童怀孕史的家庭以及有智力障碍史的家庭的畸变方面表现出了更好的表现（表。

Table 2 The diagnostic rate of different testing strategies.

表2不同测试策略的诊断率。

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Discussion

讨论

Herein, the clinical utility of OGM was investigated in families who underwent genetic consulting for infertility, recurrent miscarriages, abnormal pregnancy history with birth defects, and family history of intellectual disability. In total, 16 families were diagnosed to be positive by conventional methods.

在此，对接受不孕症，反复流产，出生缺陷异常妊娠史和智力障碍家族史遗传咨询的家庭进行了OGM的临床应用研究。通过常规方法，总共有16个家庭被诊断为阳性。

Although OGM missed one diagnosis compared to conventional methods, it diagnosed three additional aberrations in three families, improving the overall diagnostic rate by 5.4%. Furthermore, OGM could identify the abnormalities in a single test, exhibiting a high consistency with the results of CMA combined with KT (96.3%).

尽管与传统方法相比，OGM漏诊了一次，但它在三个家庭中诊断出了另外三个像差，使总体诊断率提高了5.4%。此外，OGM可以在单个测试中识别异常，与CMA联合KT（96.3%）的结果显示出高度一致性。

Among all families, OGM showed an advantage in diagnosing families with cryptic translocations, small CNVs, and complex rearrangements..

在所有家庭中，OGM在诊断隐性易位，小CNV和复杂重排的家庭方面显示出优势。。

Recently, OGM has been employed in examining genetic diseases, hematological tumors, and solid tumors

最近，OGM已被用于检查遗传疾病，血液肿瘤和实体瘤

. Additionally, various postnatal and prenatal cohort studies have proved the concordance of OGM with the combined results of CMA and KT or FISH

此外，各种产后和产前队列研究已证明OGM与CMA和KT或FISH的综合结果一致

. However, there are few studies involving different preconception genetic counseling issues. Although studies on OGM have enrolled couples with infertility or recurrent miscarriages, these studies typically have a small cohort and mainly focus on detecting cryptic balanced translocations using OGM

然而，很少有研究涉及不同的孕前遗传咨询问题。尽管对OGM的研究已经招募了不孕症或反复流产的夫妇，但这些研究通常只有一个小队列，主要集中在使用OGM检测隐性平衡易位

. Balanced translocations in couples with recurrent miscarriages can be detected by KT, but it cannot detect cryptic balanced translocations; these can be detected by OGM, making up for the lack of diagnostic ability of KT

KT可以检测到反复流产夫妇的平衡易位，但不能检测到隐性平衡易位；这些可以通过OGM检测到，弥补了KT诊断能力的不足

. Couples with a history of poor pregnancy or a family history of intellectual disability who are unable to definitively diagnose their abnormalities present various challenges in genetic counseling. Hence, in the present study, families with a pregnancy history with birth defects and a family history of intellectual disability were also included in addition to families with infertility and recurrent miscarriage.

。有不良妊娠史或智力残疾家族史的夫妇无法明确诊断其异常，在遗传咨询中面临各种挑战。因此，在本研究中，除了有不孕症和反复流产的家庭外，还包括有出生缺陷和智力残疾家族史的家庭。

The clinical indications included in the study represent the common types encountered by genetic counseling doctors in actual preconception genetic counseling. Additionally, rather than focusing on specific types of aberration, both CNVs and SVs were comprehensively evaluated in this study..

该研究中包括的临床适应症代表了遗传咨询医生在实际的孕前遗传咨询中遇到的常见类型。此外，本研究对CNV和SV进行了全面评估，而不是专注于特定类型的像差。。

Herein, the concordance rate of OGM with the conventional methods was 96.3% (Table

在此，OGM与常规方法的一致率为96.3%（表

), which is consistent with previously reported rates

)，这与先前报告的比率一致

. Furthermore, OGM showed a 5.4% increase in the diagnostic rate. Combining KT with OGM yielded the greatest diagnostic rate in families seeking preconception genetic counseling, and OGM exhibited the highest clinical diagnostic rate compared with that of KT or CMA alone. Moreover, OGM alone demonstrated a comparatively higher diagnostic rate than KT and CMA in families with a pregnancy history of children with malformations or chromosomal disorders and those with a family history of intellectual disability.

此外，OGM的诊断率提高了5.4%。在寻求孕前遗传咨询的家庭中，将KT与OGM结合使用可产生最高的诊断率，与单独使用KT或CMA相比，OGM的临床诊断率最高。此外，在有畸形或染色体疾病儿童怀孕史和有智力障碍家族史的家庭中，仅OGM的诊断率就高于KT和CMA。

Nevertheless, owing to the limited sample size, the findings of this study need to be supported by additional studies. To the best of our knowledge, no large cohort of prospective studies of families with fertility issues using OGM have been reported to date, warranting the need for such studies in the future to enhance the diagnostic rates of OGM in a more realistic context..

然而，由于样本量有限，这项研究的结果需要进一步的研究支持。据我们所知，迄今为止，尚未报道使用OGM对有生育问题的家庭进行大规模前瞻性研究，因此有必要在未来进行此类研究，以在更现实的背景下提高OGM的诊断率。。

Reportedly, OGM presents advantages in detecting cryptic translocations and complex rearrangements

. A major problem plaguing couples is when one partner is a carrier of the translocation, as it presents a high probability of reproductive failure owing to the biased formation of unbalanced gametes, typically resulting in implantation failure or abortion of the established fetus

困扰夫妇的一个主要问题是，当一个伴侣是易位的携带者时，由于配子不平衡的形成有偏差，因此很可能导致生殖失败，通常会导致植入失败或已建立的胎儿流产

. In some cases, if the unbalanced translocation is compatible with fetal development, the child may be born with a chromosomal aberration-associated disease. These findings underscore the need to detect cryptic balanced translocations. Herein, OGM additionally identified two cryptic translocations missed by KT (in families 14 and 32) and confirmed four cryptic translocations suspected by KT (Supplementary Table).

在某些情况下，如果不平衡易位与胎儿发育相适应，孩子可能出生时患有染色体畸变相关疾病。这些发现强调了检测隐性平衡易位的必要性。在此，OGM还确定了KT遗漏的两个隐性易位（在家族14和32中），并确认了KT怀疑的四个隐性易位（补充表）。

In family 32, OGM detected a cryptic translocation that disrupted .

在32族中，OGM检测到一个隐性易位，该易位被破坏。

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, which may be attributed to the development of Lessel–Kreienkamp syndrome (LESKRES), a neurodevelopmental disorder. LESKRES is characterized by the global developmental delay with intellectual disability and speech and language delays apparent from infancy or early childhood

。LESKRES的拥有属性是全球发育迟缓，智能障碍以及从婴儿期或幼儿期开始明显的言语和语言迟缓

. A study reported a patient with LESKRES presenting a heterozygous 235.3-kb deletion involving

一项研究报道，一名LESKRES患者表现出235.3 kb的杂合缺失，涉及

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and an adjacent gene

和一个相邻的基因

. Herein, the translocation was detected in intron 10 of

在此，在内含子10中检测到易位

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(located in 8q24.3), indicating that exon 1–10 was translocated to chromosome 14. Loss of function is considered to be the pathogenic mechanism of

（位于8q24.3），表明外显子1-10易位至14号染色体。功能丧失被认为是

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, suggesting that the translocation is the cause of the patient phenotype and possible implications for future pregnancies. Reportedly, OGM diagnosed a family with balanced complex rearrangements in chromosome 6, with eight breaks and fusions, including insertions and inversions. These complex rearrangements were validated by FISH, utilizing specific probes, and long-range polymerase chain reaction (LR-PCR).

，表明易位是患者表型的原因，并可能对未来怀孕产生影响。据报道，OGM诊断出一个家族在6号染色体上具有平衡的复杂重排，有八个断裂和融合，包括插入和倒位。利用特异性探针和远程聚合酶链反应（LR-PCR），通过FISH验证了这些复杂的重排。

. In some families where balanced translocations or rearrangements were detected by OGM, the parents or children of the carriers were further examined by OGM to provide a basis for future reproductive decisions for their family members. Overall, these additional findings detected by OGM significantly contributed to enhancing the diagnosis and guidance for subsequent reproduction-related decisions of these families..

。在OGM检测到平衡易位或重排的一些家庭中，OGM进一步检查了携带者的父母或孩子，为其家庭成员未来的生殖决策提供了基础。总体而言，OGM检测到的这些其他发现大大有助于增强这些家庭随后与生殖相关的决策的诊断和指导。。

The results of this study showed that OGM could detect small deletions and duplications, highlighting its high resolution. An 8-kb deletion with a potential effect on

这项研究的结果表明，OGM可以检测到小的缺失和重复，突出了其高分辨率。8 kb的缺失对

SHH

was detected in case 22OGM010 presenting twice the pregnancy history of children with HPE (Fig.

。

C). The results were verified by qPCR, confirming the deletion of exons 2 and 3 of

C）。结果通过qPCR验证，证实了外显子2和3的缺失

SHH

(Supplementary Fig. B). Consequently, this deletion was categorized as a likely pathogenic variant.

（补充图B）。因此，这种缺失被归类为可能的致病变异。

SHH

嘘

encodes a secretory protein involved in establishing cell fates at several points during development, and heterozygous mutation in

编码一种分泌蛋白，该蛋白在发育过程中的几个点参与建立细胞命运，并在

SHH

can cause HPE3. In humans, the loss of one

可能导致HPE3。在人类中，失去一个

SHH

allele is sufficient to cause HPE

等位基因足以引起HPE

, and both point mutations and microdeletions in

，以及点突变和微缺失

SHH

have been reported in fetuses with HPE

据报道，患有HPE的胎儿

. The HPE-associated

.HPE关联

SHH

mutations exhibit high heritability (73%), with 23% of parents with mutations presenting a microform

突变表现出较高的遗传力（73%），23%的突变父母表现出缩微形式

. Therefore, it was speculated that the patient included in this study might have a very mild phenotype, which when inherited in the child, made the child prone to HPE. Additionally, follow-up results showed that one of the affected children of the patient carried the deletion. The elucidation of the differential expressivity of .

因此，据推测，本研究中包括的患者可能具有非常轻微的表型，当遗传给孩子时，会使孩子容易患HPE。此外，随访结果显示，该患者的一名受影响儿童携带了该缺失。阐明的差异表达。

SHH

requires further investigations.

需要进一步调查。

Unlike CMA, OGM can clarify the location and direction of the extra copy of duplicated segments or insertions, specifying the disrupted gene. These detections may facilitate the clinical interpretation of duplications, particularly intragenic ones, during diagnosis. Herein, the relative orientation and location of three duplications in three families could be identified using OGM, compared with the results of CMA.

与CMA不同，OGM可以阐明重复片段或插入片段的额外拷贝的位置和方向，从而指定被破坏的基因。这些检测可能有助于在诊断过程中对重复，尤其是基因内重复的临床解释。在此，与CMA的结果相比，可以使用OGM识别三个家族中三个重复的相对方向和位置。

The duplications were as follows: one segmental duplication, as the translocated chromosome from the maternal parent was inherited; one Xp22.33p11.23 duplication that reversely rejoined to the Xq26.3q28 deletion (Supplementary Fig. C); and one tandem duplication in .

重复如下：一个节段重复，因为来自母本的易位染色体是遗传的；一个Xp22.33p11.23重复序列，与Xq26.3q28缺失反向重新连接（补充图C）；和一个串联重复。

DMD

DMD公司

. In family 35, a 370-kb duplication involving exon 48–56 of

在35族中，一个370Kb的重复，涉及第48-56外显子。的外显子。的研究表明，该基因的第48-56外显子与第35外显子的第48-56外显子的第48-56外显子的第48-

DMD

DMD公司

was detected in a female by CMA; however, its positional and orientational information could not be obtained by CMA. Dystrophin-associated muscular dystrophies range from the severe Duchenne muscular dystrophy (DMD) to the milder Becker muscular dystrophy (BMD)

；但是，CMA无法获得其位置和方向信息。肌营养不良蛋白相关的肌营养不良症范围从严重的杜兴氏肌营养不良症（DMD）到较轻的贝克尔肌营养不良症（BMD）

. Because both DMD and BMD are X-linked recessive diseases, the presence of the tandem duplication in the female suggests that the child of the female has a 50% chance of carrying the duplication and may affected. However, in case the duplicated segment of

由于DMD和BMD都是X连锁隐性疾病，女性中串联重复的存在表明女性的孩子有50%的机会携带重复，并可能受到影响。

DMD

DMD公司

does not occur within itself, for example, it is translocated to other chromosomes, then male carriers will not develop either DMD or BMD. Hence, obtaining the location information of the duplicated segments can help predict the clinical phenotype of the progeny of the affected individual, underscoring the potential of OGM, which could provide such information.

不会发生在自身内部，例如，它被转移到其他染色体上，那么男性携带者将不会发展DMD或BMD。因此，获得重复片段的位置信息可以帮助预测受影响个体后代的临床表型，强调OGM的潜力，它可以提供此类信息。

A recent study revealed that OGM can detect similar positional information on duplications in .

最近的一项研究表明，OGM可以检测到中重复的类似位置信息。

DMD

DMD公司

. In this case of our study, OGM detected a tandem repeat involving exons 49–56 of

在我们的研究中，OGM检测到一个串联重复序列，涉及外显子49-56

DMD

DMD公司

(Supplementary Fig. D). However, multiplex ligation-dependent probe amplification (MLPA) showed that the duplication affected exons 49–57. This could be attributed to the difference in the distribution of the CMA probe or OGM label in different

（补充图D）。然而，多重连接依赖性探针扩增（MLPA）显示重复影响外显子49-57。这可能是由于CMA探针或OGM标签在不同区域的分布不同

DMD

DMD公司

exons, which affected their accuracy in detecting duplications on both sides. Therefore, duplications involving parts of exons of

外显子，这影响了它们检测两侧重复的准确性。因此，涉及部分外显子的重复

DMD

DMD公司

need to be carefully interpreted by combining label positions on either side of the breakpoints or verified using more accurate methods such as MLPA.

需要通过结合断点两侧的标签位置仔细解释，或者使用更准确的方法（例如MLPA）进行验证。

Similar to other technologies, OGM has some limitations. OGM is unable to detect centromere fusion, single nucleotide variants, and deletions or insertions of < 500 bp. Additionally, some deletions or insertions may require verification for determining pathogenicity, requiring the employment of techniques such as LR-PCR or qPCR.

与其他技术类似，OGM也有一些局限性。OGM无法检测着丝粒融合，单核苷酸变异以及小于500 bp的缺失或插入。此外，某些缺失或插入可能需要验证以确定致病性，需要使用LR-PCR或qPCR等技术。

Nevertheless, the ability to detect variants of < 100 kb still is valuable and may be further exploited in future studies..

然而，检测小于100 kb变异的能力仍然很有价值，可能在未来的研究中进一步利用。。

Conclusions

结论

The findings of this study show that OGM technology exhibits a high concordance with conventional methods, offering notable advantages in detecting cryptic translocations and small CNVs to improve the overall diagnostic rate. Furthermore, OGM shows potential in diagnosing complex rearrangements, clarifying SVs suspected by KT, and determining the location and orientation of duplicated fragments.

这项研究的结果表明，OGM技术与常规方法具有高度一致性，在检测隐性易位和小CNV以提高总体诊断率方面具有显着优势。此外，OGM在诊断复杂的重排，澄清KT怀疑的SV以及确定重复片段的位置和方向方面显示出潜力。

The combination of OGM and KT represents a robust diagnostic strategy for families with infertility or recurrent miscarriages. In contrast, OGM alone is sufficient for diagnosing families with a pregnancy history of children with malformations or chromosomal disorders, or a family history of intellectual disability.

OGM和KT的结合代表了不孕症或反复流产家庭的强大诊断策略。相比之下，仅OGM就足以诊断有畸形或染色体疾病儿童怀孕史或智力残疾家族史的家庭。

Altogether, these findings highlight the potential of the application of OGM in clinical settings as a rapid tool to screen families seeking preconception genetic counseling. However, for prenatal diagnosis cases where precise breakpoints need to be identified, OGM alone is not sufficient and we recommend the combined use of sequencing assays..

总而言之，这些发现突显了OGM在临床环境中作为筛查寻求孕前遗传咨询的家庭的快速工具的潜力。但是，对于需要确定精确断点的产前诊断病例，仅使用OGM是不够的，我们建议联合使用测序分析。。

Materials and methods

材料和方法

Selection of individuals and collection of samples

个人选择和样本收集

In total, 74 individuals from 37 families who attended the Peking Union Medical Hospital were included in this study. The inclusion criteria were as follows: couples or families with infertility, and recurrent miscarriages; those with a pregnancy history of children with malformations, and chromosomal disorders; and those with a family history of intellectual disability.

这项研究总共包括来自北京协和医院的37个家庭的74个人。纳入标准如下：不孕夫妇或家庭，反复流产；有畸形和染色体疾病儿童怀孕史的人；以及那些有智力障碍家族史的人。

The recommended chromosomal investigations were performed per the clinical indications, such as KT and CMA, to detect SVs and submicroscopic deletions or duplications, respectively. For translocations involved in the telomere regions or special SVs, the results were validated with FISH utilizing specific probes.

推荐的染色体检查是根据临床适应症（例如KT和CMA）进行的，以分别检测SV和亚显微缺失或重复。对于涉及端粒区域或特殊SV的易位，使用特异性探针通过FISH验证了结果。

This study was conducted according to the Declaration of Helsinki principles and approved by the Ethics Committee of Peking Union Medical College Hospital (HS-2979)..

这项研究是根据赫尔辛基宣言原则进行的，并得到了北京协和医院伦理委员会（HS-2979）的批准。。

Conventional diagnostic methods

常规诊断方法

KT and CMA were performed per the standard procedures of the diagnostic laboratories. Briefly, a previously described standard protocol was followed to perform G-banded KT

KT和CMA是根据诊断实验室的标准程序进行的。简而言之，遵循先前描述的标准协议执行G带KT

. Additionally, chromosomal aberrations were described based on the International System for Human Cytogenomic Nomenclature (ISCN 2020). CMA was performed using The Affymetrix CytoScan

此外，根据国际人类细胞基因组命名系统（ISCN 2020）描述了染色体畸变。使用Affymetrix CytoScan进行CMA

750 K array (Affymetrix, Santa Clara, CA, USA) per the instructions of the manufacturer. CNVs were classified based on the guidelines of the American College of Medical Genetics and Genomics (ACMG).

750 K阵列（Affymetrix，美国加利福尼亚州圣克拉拉），按照制造商的说明。CNV是根据美国医学遗传学和基因组学学院（ACMG）的指南进行分类的。

Sample processing for OGM

OGM样品处理

The Bionano Prep SP Frozen Human Blood DNA Isolation Protocol v2 (Bionano Genomics, San Diego, CA, USA) was used to extract ultra-high-molecular-weight (UHMW) DNA from whole blood or amniotic fluid cells using the SP Blood and Cell Culture DNA Isolation Kit (Bionano Genomics, San Diego, USA,80042). When the concentration of UHMW DNA was 36–150 ng/µL, the Direct Label and Stain kit (Bionano Genomics) was used per the protocol of the manufacturer to label the CTTAAG motif on 750 ng of DNA (green fluorescence), and the DNA backbone was counterstained.

使用Bionano Prep SP冷冻人血DNA分离方案v2（Bionano Genomics，圣地亚哥，加利福尼亚，美国）使用SP血液和细胞培养DNA分离试剂盒从全血或羊水细胞中提取超高分子量（UHMW）DNA。（Bionano Genomics，圣地亚哥，美国，80042）。当UHMW DNA的浓度为36–150 ng/µL时，根据制造商的协议使用直接标记和染色试剂盒（Bionano Genomics）在750 ng DNA（绿色荧光）上标记CTTAAG基序，并对DNA骨架进行复染。

Qubit dsDNA HS Assay Kit and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to quantify the labeled DNA to achieve a concentration of 4–12 ng/µL, following which, the labeled DNA was loaded on a Saphyr chip (Bionano Genomics)..

使用Qubit dsDNA HS测定试剂盒和Qubit荧光计（Thermo Fisher Scientific，Waltham，MA，USA）对标记的DNA进行定量，以达到4-12 ng/µL的浓度，然后将标记的DNA加载到Saphyr芯片（Bionano Genomics）上。。

Quality control and data analyses for OGM

OGM的质量控制和数据分析

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

使用Saphyr仪器（Bionano）对芯片上加载的DNA进行成像和拍照，并将图像转换为数字数据以获得DNA序列。建立了以下四个主要质量控制参数：分子N50（≥150 kb）>230 kb，每100 kb的平均标记密度（≥150 kb）为14-17，分子到参考基因组的映射率≥70%，有效基因组覆盖率≥120倍。。

Variant calling and filtering for OGM

OGM的变体调用和过滤

Herein, Bionano Solve™ (v3.7) and OGM-specific pipelines managed by Bionano Access™ (v1.7) were used to analyze the SVs and CNVs in the genome by assembling molecules into consensus maps using the de novo pipeline. SVs were identified by comparing the sample consensus maps with the reference genome (hg19), and a copy number algorithm was used to detect CNVs.

。通过将样本共有图谱与参考基因组（hg19）进行比较来鉴定SV，并使用拷贝数算法检测CNV。

Following this, both CNVs and SVs were filtered according to the following parameters: (1) SVs ≤ 1500 bp; (2) SV frequency in the built-in control database < 1%; (3) minimum size of CNV ≥ 200 kb; and (4) SVs overlapping genes within 3 kb precision of the GRCh19 known canonical gene breakpoints. Filtered SVs were then analyzed and interpreted per the ACMG guidelines..

在此之后，根据以下参数过滤CNV和SV：（1）SV≤1500 bp；（2）内置控制数据库中的SV频率<1%；（3） CNV的最小大小≥200 kb；（4）SVs重叠基因在GRCh19已知规范基因断点的3 kb精度内。然后根据ACMG指南对过滤后的SV进行分析和解释。。

Verification methods

验证方法

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

对鱼进行了标准程序，包括载玻片制备，杂交，杂交后洗涤和复染。对于LR-PCR，根据制造商（Toyobo，大阪，日本）的说明扩增断点区域。此后，进行Sanger测序以进一步验证目标区域的确切融合位点。。

Data availability

数据可用性

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

本研究中使用和/或分析的数据集可根据合理要求从通讯作者处获得。

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Acknowledgements

致谢

The authors appreciate the individuals and their families for their participation in this study.

作者感谢个人及其家人参与这项研究。

Funding

资金

The research was supported by the National High Level Hospital Clinical Research (2022-PUMCH-B-076) and the CAMS Innovation Fund for Medical Sciences (CIFMS-2022-I2M-C&T-B-008).

该研究得到了国家高水平医院临床研究（2022-PUMCH-B-076）和CAMS医学科学创新基金（CIFMS-2022-I2M-C＆T-B-008）的支持。

Author information

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Authors and Affiliations

作者和隶属关系

National Clinical Research Center for Obstetric & Gynecologic Diseases, Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China

中国医学科学院北京协和医科大学附属北京协和医院妇产科国家妇产科临床研究中心

Kaili Yin, Mengmeng Li, Hanzhe Zhang, Jiazhen Chang, Qingwei Qi, Xiya Zhou, Na Hao & Yulin Jiang

尹凯丽、李梦萌、张汉哲、常佳珍、齐庆伟、周希亚、Na Hao和姜玉林

Becreative Lab Co., Ltd, Beijing, China

Becreative Lab Co.，Ltd，中国北京

Jiangshan Guo

郭江山

Ecobono (Beijing) Biotech Co., Ltd, Beijing, China

爱科博诺（北京）生物技术有限公司，中国北京

Yaru Wang

王亚如

Haidian District Maternal and Child Health Care Hospital, Beijing, China

中国北京市海淀区妇幼保健院

Xuequn Mao

毛游戏

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Kaili Yin

凯里印

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Mengmeng Li

李孟

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Hanzhe Zhang

张汉哲

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王亚如

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Contributions

捐款

N.H. coordinated the project and designed the experiment, and revised the manuscript. K.Y. participated in the data analysis and drafted the manuscript. M.L., H.Z., J.C., Y.W., J.G., and X.M. executed different parts of the research. Q.Q. and X.Z. directed the critical discussion of the manuscript. Y.J.

N、 H.协调项目并设计实验，并修改手稿。K、。M、 L.，H.Z.，J.C.，Y.W.，J.G。和X.M.执行了研究的不同部分。Q、 Q.和X.Z.指导了手稿的批判性讨论。Y、 J。

conceived and revised the paper. All authors approved the final manuscript..

构思并修改了论文。所有作者都批准了最终稿件。。

Corresponding authors

通讯作者

Correspondence to

通信对象

Na Hao

纳浩

或

Yulin Jiang

江玉林

Ethics declarations

道德宣言

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Ethical approval

道德认可

This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Peking Union Medical College Hospital (HS-2979). All study participants consented to undergo genetic evaluations and signed written informed consent.

这项研究是根据《赫尔辛基宣言》的原则进行的，并得到了北京协和医院伦理委员会（HS-2979）的批准。所有研究参与者均同意接受基因评估并签署书面知情同意书。

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Electronic supplementary material

电子补充材料

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以下是电子补充材料的链接。

Supplementary Material 1

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Supplementary Material 2

补充材料2

Supplementary Material 3

补充材料3

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Yin, K., Li, M., Zhang, H.

尹K.，李M.，张H。

et al.