Liu Ying, Lei Cheng, Wang Rongchun, Yang Danhui, Yang Binyi, Xu Yingjie, Lu Chenyang, Wang Lin, Ding Shuizi, Guo Ting, Liu Shaokun, Luo Hong
Department of Pulmonary and Critical Care Medicine, The Second Xiangya Hospital, Central South University, Changsha, China.
Research Unit of Respiratory Disease, Central South University, Changsha, China.
Front Genet. 2022 Jul 6;13:940292. doi: 10.3389/fgene.2022.940292. eCollection 2022.
Whole-exome sequencing (WES) based copy number variation (CNV) analysis has been reported to improve the diagnostic rate in rare genetic diseases. In this study, we aim to find the disease-associated variants in a highly suspected primary ciliary dyskinesia (PCD) patient without a genetic diagnosis by routine WES analysis. We identified the CNVs using the "Exomedepth" package in an undiagnosed PCD patient with a negative result through routine WES analysis. RNA isolation, PCR amplification, and Sanger sequencing were used to confirm the variant. High-speed video microscopy analysis (HSVA) and immunofluorescence analysis were applied to detect the functional and structural deficiency of nasal cilia and sperm flagella. Papanicolaou staining was employed to characterize the morphology of sperm flagella. NC_000002.11(NM_145038.5): g.26635488_26641606del, c.156-1724_244-2550del, r.156_243del, p. (Glu53Asnfs*13), a novel homozygous CNV, was identified by WES-based CNV analysis rather than routine variants calling, in a patient from a non-consanguineous family. HSVA results showed no significant change in ciliary beating frequency but with reduced beating amplitude compared with normal control, and his spermatozoa were almost immotile. The diagnosis of multiple morphological abnormalities of the sperm flagella (MMAF) was established through sperm motility and morphology analysis. PCR amplification and Sanger sequencing confirmed the novel variant of . Immunofluorescence showed that both cilia and sperm flagella were deficient in protein expression related to the dynein regulatory complex. This report identifies a novel disease-associated variant by WES-based CNV analysis from a highly suspected PCD patient with MMAF. Our findings not only expand the genetic spectrum of PCD with MMAF but suggest that in combination with CNV analysis might improve the efficiency of genetic tests.
据报道，基于全外显子组测序（WES）的拷贝数变异（CNV）分析可提高罕见遗传病的诊断率。在本研究中，我们旨在通过常规WES分析在一名高度疑似原发性纤毛运动障碍（PCD）但未确诊的患者中找到与疾病相关的变异。我们在一名通过常规WES分析结果为阴性的未确诊PCD患者中使用“Exomedepth”软件包鉴定CNV。采用RNA分离、PCR扩增和Sanger测序来确认变异。应用高速视频显微镜分析（HSVA）和免疫荧光分析来检测鼻纤毛和精子鞭毛的功能和结构缺陷。采用巴氏染色来表征精子鞭毛的形态。通过基于WES的CNV分析而非常规变异检测，在一个非近亲家庭的患者中鉴定出一个新的纯合CNV：NC_000002.11（NM_145038.5）：g.26635488_26641606del，c.156 - 1724_244 - 2550del，r.156_243del，p.（Glu53Asnfs*13）。HSVA结果显示，与正常对照相比，纤毛摆动频率无显著变化，但摆动幅度降低，且其精子几乎无运动能力。通过精子活力和形态分析确诊为精子鞭毛多发形态异常（MMAF）。PCR扩增和Sanger测序证实了该新变异。免疫荧光显示，纤毛和精子鞭毛中与动力蛋白调节复合体相关的蛋白表达均缺失。本报告通过基于WES的CNV分析从一名高度疑似PCD且患有MMAF的患者中鉴定出一个新的疾病相关变异。我们的发现不仅扩展了伴有MMAF的PCD的遗传谱，还表明结合CNV分析可能提高基因检测的效率。
