在中国进行低水平母体性腺嵌合体的Cornelia de Lange综合征亚大碱基缺失的植入前遗传学检测

摘要(中文译文)

为对一个受科妮莉亚·德·兰格综合征5型（Cornelia de Lange syndrome type 5）影响的中国家系进行诊断和单基因病植入前遗传学检测（PGT-M），该家系由Xq13.1q13.2微缺失导致，该缺失截断相关基因，并以低水平性腺嵌合体为特征。通过染色体微阵列分析和全外显子组测序，在一个有两次妊娠失败史的家系中鉴定出一处新发拷贝数变异（CNV），提示生殖系嵌合体。该CNV通过实时定量PCR得到验证。对女性外周血进行全基因组低覆盖度配对末端测序，以排除染色体异常。利用长PCR扩增缺失片段，所用引物设计于通过染色体微阵列分析鉴定出的断点附近。Oxford Nanopore Technology测序精确定位了具体断点位置。微滴数字PCR（ddPCR）证实了卵巢样本中的生殖系嵌合体。一名疑似携带Xq13.1q13.2新发微缺失并表现为性腺嵌合体的女性患者及其丈夫参与了本研究。通过纳米孔测序和ddPCR证实了Xq13.1q13.2处172.3 kb微缺失为低水平性腺嵌合体的诊断。我们以受累的流产妊娠产物作为定相参考构建了高风险单倍型，并基于SNP单倍型连锁分析实施PGT-M，最终于2023年2月获得健康活产。我们的发现强调了采用单倍型连锁分析的PGT-M对于小于1 Mb的CNV的有效性，即使在涉及性腺嵌合体的病例中亦然。我们提出了利用下一代测序、微阵列、纳米孔测序和ddPCR技术处理与性腺嵌合体相关微缺失的方法。我们的结果支持将基于单倍型连锁分析的PGT-M扩展应用于携带微小致病性CNV的家系。

Abstract

To diagnose and perform preimplantation genetic testing for monogenic disorders (PGT-M) in a Chinese family affected by Cornelia de Lange syndrome type 5, resulting from a microdeletion in Xq13.1q13.2 truncating the gene characterized by low-level gonadal mosaicism. A de novo copy number variation (CNV) was identified through chromosomal microarray analysis and whole exome sequencing in a family with two unsuccessful pregnancies, indicating germline mosaicism. The CNV was validated via real-time quantitative PCR. Whole-genome low-coverage mate-pair sequencing was conducted on female peripheral blood to exclude chromosomal abnormalities. Long-PCR amplified the deleted fragment, utilizing primers designed nearby breakpoints identified through chromosomal microarray analysis. Oxford Nanopore Technology sequencing pinpointed specific breakpoint positions. Droplet-digital PCR (ddPCR) confirmed germline mosaicism in ovarian samples. A female patient, suspected of harboring a de novo microdeletion at Xq13.1q13.2 exhibiting gonadal mosaicism, along with her husband, participated in this study. The diagnoses of the 172.3 kb microdeletion at Xq13.1q13.2 as low-level gonadal mosaicism was corroborated through nanopore sequencing and ddPCR. We constructed the high-risk haplotype using the affected products of conception as the phasing reference, and performed PGT-M based on SNP haplotype linkage analysis, finally achieving a healthy live birth in February 2023. Our findings underscore the efficacy of PGT-M employing haplotype linkage analysis for CNVs less than 1 Mb, even within cases involving gonadal mosaicism. We present methodologies to address microdeletions associated with gonadal mosaicism utilizing next-generation sequencing, microarray, nanopore sequencing and ddPCR techniques. Our results advocate for an expansion of PGT-M based on haplotype linkage analysis for families with minor pathogenic CNVs.

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