Data Sheet 1_Analysis of complex chromosomal structural variants through optical genome mapping integrated with karyotyping.docx

Background and ObjectiveParental chromosomal structural variations (SVs) represent a primary genetic factor contributing to recurrent spontaneous abortion (RSA). Individuals carrying SVs with complex chromosomal rearrangements (CCRs) typically exhibit a normal phenotype but are at an increased risk of miscarriage. Current standard clinical detection methods are insufficient for the identification and interpretation of all SV types, particularly complex and occult SVs, thereby presenting a significant challenge for clinical genetic counseling. Leveraging the high-resolution capabilities of optical genome mapping (OGM) technology, this study aims to rapidly and accurately identify complex SVs in RSA couples. Furthermore, it seeks to conduct an in-depth analysis of the genetic information within the breakpoint regions, thereby providing a more comprehensive scientific foundation for genetic counseling of RSA couples at both the cellular and genetic levels.
Material and MethodsThis study involved the selection of nine subjects from two families who underwent genetic counseling at our hospital. Family 1 comprised a couple with the wife as a SVs carrier, and both her parents and brother were simultaneously analyzed for chromosomal karyotype. Family 2 included a couple with the husband as the SVs carrier, with his parents also undergoing chromosomal karyotype analysis. For SVs carriers whose karyotype analysis did not elucidate the recombination pattern, optical genome mapping (OGM) technology was utilized for further investigation, followed by Sanger sequencing to validate the OGM findings.
ResultsIn Family 1, only the wife was identified as an SVs carrier. Initial chromosomal karyotype analysis suggested a karyotype of 46,XX,t (5; 6;8; 13; 15) (?). However, OGM analysis ultimately confirmed the karyotype as 46,XY,der (5)t (5; 13) (q35.2; q21.32), der (6)t (6; 8) (q25.3; q13.1)ins (6; 13) (q25.3; q21.32q21.33),der (8)t (6; 8) (q26; q13.1)ins (8; 13) (q13.1; q21.33q22.1),der (13)t (13; 15) (q21.32; q26.1)ins (13; 6) (q21.32; q25.3q26), der (15)t (5; 15) (q35.2; q26.1). Furthermore, OGM identified a novel translocation variant of the KIF7 gene that is associated with recurrent miscarriage. In Family 2, both the husband and his maternal parent were identified as SVs carriers. Nuclear type analysis revealed a karyotype of 46,XY,?t (1; 6) (q42; p21) (husband) and 46,XX,?t (1; 2) (p31.1; q24.1),?t (1; 6) (q42; p21) (mother). Through OGM detection and analysis, the final karyotype was determined to be 46,XY,ins (1; 6) (q42.2; p22.3p11.3) (husband) and 46,XX,der (1)t (1; 2) (p31.1; q24.1)ins (1; 6) (q42.2; p22.3p11.3), der (2) t (1; 2), der (6)ins (1; 6) (mother).
ConclusionOGM technology facilitates the rapid and precise identification of complex chromosomal structural variations, effectively overcoming the limitations associated with traditional karyotype G-banding techniques in detecting intricate and cryptic SVs. This advancement substantially enhances the diagnostic rates of genetic etiology in patients experiencing RSA. The present study elucidates the specific manifestations of complex SVs using OGM technology, accurately pinpointing breakpoints and interpreting affected gene information. This provides novel reference approaches and evidence for disease assessment and genetic counseling in RSA patients. However, it is important to acknowledge certain limitations of this research: the study’s inclusion of only two RSA family cohorts (comprising nine participants) may limit the generalizability of its conclusions due to the small sample size, necessitating further validation through large-scale studies. Additionally, the causal relationship between KIF7 gene dysfunction and recurrent miscarriage remains to be experimentally verified in subsequent research.

背景与目的：父母源性染色体结构变异（chromosomal structural variations, SVs）是导致复发性自然流产（recurrent spontaneous abortion, RSA）的主要遗传学因素之一。携带复杂染色体重排（complex chromosomal rearrangements, CCRs）型SVs的个体通常表型正常，但流产风险显著升高。当前临床标准检测方法无法全面识别并解析所有类型的SVs，尤其是复杂隐匿性SVs，这为临床遗传咨询带来了极大挑战。本研究借助光学基因组图谱（optical genome mapping, OGM）技术的高分辨率优势，旨在快速精准地识别复发性自然流产夫妇体内的复杂SVs，并深入分析断裂点区域的遗传信息，从而为复发性自然流产夫妇的细胞与遗传学层面的遗传咨询提供更全面的科学依据。

材料与方法：本研究纳入于我院接受遗传咨询的两个家庭共9名研究对象。家系1为妻子携带SVs的复发性自然流产夫妇，同时对其父母及兄弟进行染色体核型分析。家系2为丈夫携带SVs的复发性自然流产夫妇，同时对其父母开展染色体核型分析。对于核型分析未能明确重组模式的SVs携带者，采用光学基因组图谱（OGM）技术进行进一步检测，随后通过Sanger测序验证OGM的检测结果。

结果：家系1中仅妻子被鉴定为SVs携带者。初始染色体核型分析提示核型为46,XX,t(5;6;8;13;15)(?)，但经OGM分析最终确认其核型为46,XY,der(5)t(5;13)(q35.2;q21.32)、der(6)t(6;8)(q25.3;q13.1)ins(6;13)(q25.3;q21.32q21.33)、der(8)t(6;8)(q26;q13.1)ins(8;13)(q13.1;q21.33q22.1)、der(13)t(13;15)(q21.32;q26.1)ins(13;6)(q21.32;q25.3q26)以及der(15)t(5;15)(q35.2;q26.1)。此外，OGM还检测到1个与复发性流产相关的KIF7基因新型易位变异体。家系2中，丈夫及其母亲均被鉴定为SVs携带者。核型分析初步提示丈夫核型为46,XY,?t(1;6)(q42;p21)，其母亲核型为46,XX,?t(1;2)(p31.1;q24.1)、?t(1;6)(q42;p21)。经OGM检测与分析后，最终确定丈夫核型为46,XY,ins(1;6)(q42.2;p22.3p11.3)，其母亲核型为46,XX,der(1)t(1;2)(p31.1;q24.1)ins(1;6)(q42.2;p22.3p11.3)、der(2)t(1;2)、der(6)ins(1;6)。

结论：OGM技术可快速精准地识别复杂染色体结构变异，有效克服了传统G显带核型分析技术在检测复杂隐匿性SVs时的局限性，显著提升了复发性自然流产患者的遗传病因诊断率。本研究借助OGM技术阐明了复杂SVs的具体表现形式，精准定位断裂点并解析受累基因信息，为复发性自然流产患者的病情评估与遗传咨询提供了全新的参考方法与依据。但本研究仍存在一定局限性：仅纳入2个复发性自然流产家系（共9名研究对象），样本量较小可能限制结论的外推性，需后续开展大规模研究进一步验证；此外，KIF7基因功能异常与复发性流产之间的因果关系仍需后续实验研究予以证实。