Malone Andrew F, Funk Steven D, Alhamad Tarek, Miner Jeffrey H
Division of Nephrology, Department of Medicine, Washington University School of Medicine, 4523 Clayton Avenue, St. Louis, MO, 63110, USA.
Pediatr Nephrol. 2017 Jun;32(6):997-1003. doi: 10.1007/s00467-016-3565-4. Epub 2016 Dec 24.
Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis.
Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy.
A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing.
Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.
在X连锁遗传性肾炎患者中已描述了许多COL4A5剪接区域变异，但经功能分析证实实际导致剪接缺陷的却很少。我们试图通过功能研究证明一个遗传性肾炎家族中的新型COL4A5剪接区域变异具有致病性。我们还描述了一种替代诊断方法。
对一名遗传性肾炎患者的靶向二代测序结果进行分析，并通过桑格测序法在家族成员中进行结果确认。使用剪接报告子小基因检测法检测该变异对转染细胞剪接的影响。利用免疫荧光显微镜检查患者和对照者拔下的毛囊中的IV型胶原蛋白。
在该家族中鉴定出COL4A5的一个新型剪接区域突变c.1780-6T>G，并与疾病共分离。该变异导致外显子25频繁跳跃，导致胶原蛋白α5(IV)蛋白发生移码和截短。我们还开发并验证了一种新方法，用于表征拔下毛囊基底膜中胶原蛋白α5(IV)蛋白的表达。使用这种方法，我们证明了该家族中受影响的男性和女性个体的胶原蛋白α5(IV)蛋白减少，支持正常剪接经常失败。
携带剪接区域变异的受影响个体中正常转录本与异常转录本比例的差异可能导致遗传性肾炎家族中观察到的疾病严重程度不同。对疑似X连锁遗传性肾炎患者的拔下毛囊进行检查可能提供一种侵入性较小的替代诊断方法，并作为对意义不确定的COL4A5变异的致病性检测。
