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

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.

Keywords: Alport syndrome; COL4A5; Hair follicle; Immunofluorescence; Splice site mutation.

Figure 1. Pedigree of Alport Family 68 with chromatograms showing that the c.1780-6T>G splice region variant is present in affected but not unaffected family members

Squares, males; circles, females; open shapes, unaffected; filled shapes, affected; diagonal strike, deceased. The numbers beneath the icons are individual identification numbers and correspond to the respective chromatograms. The heterozygous variant in females shown as overlapping peaks in the chromatograms is denoted by brackets.

Figure 2. In vitro analysis of the splice region variant’s function in HEK293T cells reveals frequent skipping of exon 25

(A) The diagram illustrates the relevant intronic (dashed lines) and exonic (boxes) regions of the splicing reporter minigene vector. Black boxes represent C1NH exons 2 and 3, and the open box represents exon 25 of COL4A5. The c.1780-6T>G splice region variant is indicated. (B) RT-PCR products obtained from cells transfected with wild type (wt) vector only, variant (var) vector only, or wt and var vectors together. Product sizes are indicated based on size markers in lane 4. Diagrams to the left represent the normally spliced transcript containing exon 25 (top) and the abnormally spliced transcript resulting from the c.1780-6T>G variant (bottom).

Figure 3. Immunofluorescence staining of plucked hair follicle basement membranes from a control and from Alport family members

FITC-conjugated antibodies to collagen α5(IV) (green) and Texas Red-conjugated antibody to collagen α2(IV) (red) were used. A, Control follicle, COL4A5^WT/WT. B, 3223 follicle, COL4A5^{c.1780-6T>G/WT}. C, 3227 follicle, COL4A5^{c.1780-6T>G/Y}. D, 3231 follicle, COL4A5^WT/Y. COL4A2 staining shows where basement membrane is present. COL4A5 was abundant in the control (A) and unaffected family member (D) follicle basement membranes but absent (B) or weak/segmental (C) in those with the variant.