Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.
Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.
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DOI：
10.1007/s00467-016-3565-4
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发表时间：
2017-06
期刊：
影响因子：
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通讯作者：
Miner JH
中科院分区：
文献类型：
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作者：
Malone AF;Funk SD;Alhamad T;Miner JH
在X连锁Alport综合征患者中已经描述了许多COL 4A 5剪接区变体，但很少有功能分析证实实际上导致剪接缺陷。我们试图证明一个新的COL 4A 5剪接区变异在一个家庭与Alport综合征是致病性的功能研究。我们还描述了一种替代的诊断方法。我们分析了Alport综合征个体的靶向下一代测序结果，并通过家族成员的桑格测序证实了结果。剪接报告基因小基因测定用于检查变体对转染细胞中剪接的影响。采用免疫荧光显微镜检查患者和对照组的毛囊中的IV型胶原蛋白。在该家系中发现了一种新的COL 4A 5剪接区突变，c.1780- 6 T>G，并与疾病分离。这种变异导致外显子25的频繁跳跃，导致胶原α5（IV）蛋白的移码和截短。我们还开发并验证了一种新的方法来表征胶原α5（IV）蛋白在拔出的毛囊基底膜中的表达。我们证明了在这个家族中受影响的男性和女性个体中胶原α5（IV）蛋白的减少，支持正常剪接的频繁失败。不同的正常异常转录比受影响的个人携带剪接区变异体可能有助于可变的疾病严重程度观察Alport家庭。在疑似X连锁Alport综合征患者中检查拔出的毛囊可能提供一种侵入性较小的替代诊断方法，并可作为意义不确定的COL 4A 5变体的致病性试验。
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. We analyzed targeted next-generation sequencing results of an individual with Alport syndrome and confirmed results 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. 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.