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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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Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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Acknowledgements

We thank Diane Salamon in the Division of Nephrology’s Kidney Translational Research Core for processing human specimens, Gloriosa Go for technical assistance, and Dr. Mario Tosi (Inserm U1079, Rouen, France) for generously providing the pCAS2.1 splicing vector. JHM was supported by NIH grants R56DK100593, R01DK078314, and R01DK058366 and by the Alport Syndrome Foundation/Pedersen Family/Kidney Foundation of Canada Alport Syndrome Research Funding Program. SDF was supported by T32DK007126.

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Authors and Affiliations

  1. Division of Nephrology, Department of Medicine, Washington University School of Medicine, 4523 Clayton Avenue, St. Louis, MO, 63110, USA

    Andrew F. Malone, Steven D. Funk, Tarek Alhamad & Jeffrey H. Miner

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  1. Andrew F. Malone
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  2. Steven D. Funk
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  3. Tarek Alhamad
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Correspondence to Jeffrey H. Miner.

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Ethics statement

Human studies were conducted according to protocols approved by the Washington University Human Research Protection Office. Informed consent was obtained from all individual participants included in the study.

Disclosures

JHM has received grants from Hoffmann-La Roche, Basel and RGDI3, Inc., Boston; has provided consultation to Third Rock Ventures, Boston; and has received licensing fees from Eli Lilly, Indianapolis and Genentech, South San Francisco. The other authors declare that they have no conflicts of interest

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Malone, A.F., Funk, S.D., Alhamad, T. et al. 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. Pediatr Nephrol 32, 997–1003 (2017). https://doi.org/10.1007/s00467-016-3565-4

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  • DOI: https://doi.org/10.1007/s00467-016-3565-4

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