Bestetti Ilaria, Crippa Milena, Sironi Alessandra, Bellini Matteo, Tumiatti Francesca, Ballabio Sara, Ceriotti Ferruccio, Memo Luigi, Iascone Maria, Larizza Lidia, Finelli Palma
SC Patologia Clinica, SS Laboratorio Genetica Medica, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy.
Laboratorio Sperimentale di Ricerche di Citogenetica Medica e Genetica Molecolare, IRCCS Istituto Auxologico Italiano, Milano, Italy.
Front Genet. 2024 Mar 13;15:1358334. doi: 10.3389/fgene.2024.1358334. eCollection 2024.
Thanks to a long-read sequencing (LRS) approach, in this study, we have reported a molecularly solved case of a proband with a clinical diagnosis of Cornelia de Lange syndrome (CDLS), which is a multisystemic disorder whose causative molecular defects involve cohesin complex genes, with located at 5p13.2 accounting for approximately 50%-60% of CDLS cases. The first-tier tests revealed an abnormal karyotype 46,XY,t(5;15)(p13;q25)dn and a preserved sequencing. Copy number variants (CNVs) at the translocation breakpoints, in disease genes, or in probably pathogenic loci were excluded by a-CGH analysis. Through fluorescence hybridization (FISH) analysis on derivative chromosome 5, the breakpoint was relocated 3 Mb far from 5'UTR, which seemed fully maintained as FISH-probe mapping to the gene showed no split signals. Moreover, tri-color FISH revealed an apparently balanced paracentric inversion including on derivative 5. Based on the strong clinical suspicion, we evaluated the transcript by RT-qPCR that revealed a normal amount of transcript till exon 22 and a halved amount of the transcript from exon 23 to 3'UTR, indicating the expression of a truncated transcript probably leading to a defective protein. Despite RT-qPCR confirmed the patient's CDLS clinical diagnosis, the molecular mechanism underlying this event remained to be an unsolved challenge for years. The LRS approach with nanopore technologies was able to fill the gap in this complex scenario and highlighted a chromothripsis event marked out at 5p13.2 by 36 breaks clustered in a 7.3-Mb region. The gene was disrupted by 16 breaks and the resulting fragments were relocated in different positions and orientations. LRS confirmed the previous findings, and it has been proven to be crucial to define the complex chromosomal rearrangement in this patient which escaped current diagnostic investigations. Its application in the clinical practice will contribute to solve the unsolved.
得益于长读长测序（LRS）方法，在本研究中，我们报告了一例临床诊断为科妮莉亚·德·朗格综合征（CDLS）的先证者分子水平确诊病例。CDLS是一种多系统疾病，其致病分子缺陷涉及黏连蛋白复合体基因，位于5p13.2的基因约占CDLS病例的50%-60%。一级检测显示核型异常为46,XY,t(5;15)(p13;q25)dn且测序结果正常。通过a-CGH分析排除了易位断点处、疾病基因中或可能的致病位点的拷贝数变异（CNV）。通过对衍生5号染色体的荧光原位杂交（FISH）分析，断点重新定位到距离基因5'UTR 3 Mb处，由于映射到该基因的FISH探针未显示分裂信号，该基因似乎完全保持完整。此外，三色FISH显示衍生5号染色体上存在一个明显平衡的臂内倒位，包括该基因。基于强烈的临床怀疑，我们通过RT-qPCR评估该基因转录本，结果显示直到外显子22转录本数量正常，而从外显子23到3'UTR转录本数量减半，表明存在截短转录本的表达，可能导致蛋白质缺陷。尽管RT-qPCR证实了患者的CDLS临床诊断，但多年来该事件背后的分子机制仍是一个未解之谜。采用纳米孔技术的LRS方法能够填补这一复杂情况下的空白，并突出显示了在5p13.2处发生的染色体碎裂事件，36个断点聚集在一个7.3-Mb区域。该基因被16个断点破坏，产生的片段重新定位到不同的位置和方向。LRS证实了先前的发现，并且已证明对于确定该患者复杂的染色体重排至关重要，而这种重排逃过了当前的诊断研究。其在临床实践中的应用将有助于解决未解之谜。
