PDF(Pubmed)
Abstract：
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 NIPBL 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 NIPBL 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 in situ hybridization (FISH) analysis on derivative chromosome 5, the breakpoint was relocated 3 Mb far from NIPBL 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 NIPBL on derivative 5. Based on the strong clinical suspicion, we evaluated the NIPBL 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 NIPBL 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.
摘要：
由于采用了长读数测序（LRS）方法，在这项研究中，我们报道了一个分子解决的病例，临床诊断为CorneliadeLange综合征（CDLS），这是一种多系统疾病，其致病分子缺陷涉及复杂的基因，NIPBL位于5p13.2，约占CDLS病例的50％-60％。第一层测试显示异常核型46，XY，t(5;15)(p13;q25)dn和保留的NIPBL测序。易位断点处的拷贝数变体（CNV），在疾病基因中，通过a-CGH分析排除或可能的致病位点。通过对衍生染色体5的荧光原位杂交（FISH）分析，断点被重新定位为3Mb，远离NIPBL5'UTR，这似乎完全维持，因为FISH探针对基因的定位没有显示分裂信号。此外，三色FISH显示出明显平衡的副中心反转，包括导数5上的NIPBL。基于强烈的临床怀疑，我们通过RT-qPCR评估了NIPBL转录本，该转录本揭示了正常量的转录本直到外显子22和一半量的转录本从外显子23到3'UTR，表明截短的转录物的表达可能导致缺陷蛋白。尽管RT-qPCR证实了患者的CDLS临床诊断，这一事件背后的分子机制多年来仍是一个未解决的挑战.具有纳米孔技术的LRS方法能够填补该复杂场景中的空白，并突出显示了在5p13.2处标记为7.3-Mb区域中聚集36个断裂的色素沉着事件。通过16个断裂破坏NIPBL基因,并且将所得片段重新定位在不同的位置和取向。LRS证实了之前的发现，并且已经证明，在该患者中定义复杂的染色体重排是至关重要的，该患者逃脱了当前的诊断研究。其在临床实践中的应用将有助于解决尚未解决的问题。
