TNXB mutations can cause vesicoureteral reflux

Abstract

Primary vesicoureteral reflux (VUR) is the most common congenital anomaly of the kidney and the urinary tract, and it is a major risk factor for pyelonephritic scarring and CKD in children. Although twin studies support the heritability of VUR, specific genetic causes remain elusive. We performed a sequential genome-wide linkage study and whole-exome sequencing in a family with hereditary VUR. We obtained a significant multipoint parametric logarithm of odds score of 3.3 on chromosome 6p, and whole-exome sequencing identified a deleterious heterozygous mutation (T3257I) in the gene encoding tenascin XB (TNXB in 6p21.3). This mutation segregated with disease in the affected family as well as with a pathogenic G1331R change in another family. Fibroblast cell lines carrying the T3257I mutation exhibited a reduction in both cell motility and phosphorylated focal adhesion kinase expression, suggesting a defect in the focal adhesions that link the cell cytoplasm to the extracellular matrix. Immunohistochemical studies revealed that the human uroepithelial lining of the ureterovesical junction expresses TNXB, suggesting that TNXB may be important for generating tensile forces that close the ureterovesical junction during voiding. Taken together, these results suggest that mutations in TNXB can cause hereditary VUR.

Figure 1.

GWLSs in a family with hereditary VUR. (A) Abbreviated pedigree of a 96-member kindred with hereditary VUR. There are at least nine affected individuals with imaging confirmation of VUR/duplex kidney. Squares are male family members and circles are female family members. Obligate heterozygous individuals are shown in partially filled circle/rectangle. (B) Imaging from one of the affected individuals showing grade 4 VUR on the left side. DMSA scan from the same individual showed left renal parenchymal scarring. (C) Genome-wide linkage scan using the Illumina Infinum II HumanLinkage-24 genotyping beadchip assay yields multipoint LOD scores of 3.3 on chromosome 6p in the family. (D) Fine mapping of the chromosome 6p locus and haplotype of individuals defining the chromosome 6P minimal candidate region. Individual 1001 has the ancestral haplotype, the centromeric flanking marker D6S109 is defined by affected individual 1, and telomeric flanking marker D6S271 defined by affected individual 103.

Figure 2.

Missense mutations in TNXB as a cause of VUR. (A) Exons and protein domains of TNXB. (B and C) Missense heterozygous mutation exon 29. 9770 C>T T3257I found in the index family with hereditary VUR; this mutation is conserved in evolution. (D and E) Another kindred with VUR was found to have G1331R mutation in exon 10; this mutation is also conserved in evolution.

Figure 3.

Reduced cell motility and expression of phosphorylated FAK in a fibroblast cell line of an individual with T3257I mutation. (A) Representative images of WT and mutant dermal fibroblast wound healing assays in the presence of PDGF versus vehicle alone. Note the attenuation of motility in TNXB mutant-expressing fibroblasts (Mut T3257I) in response to PDGF relative to the WT cell line. (B) The results of three individual wound healing assays expressed as percent wound healing are quantified. (C) Representative images of immunoblots from WT and mutant fibroblasts with staining for TNXB, FAK, and pY397-FAK. Note that TNXB expression is similar in all groups and that the PDGF-induced phosphorylation of FAK at tyrosine 397 is significantly attenuated in mutant (Mut T3257I) versus the WT fibroblast cell line.

Figure 4.

TNXB is expressed in the UVJ of normal and refluxing ureters. Human UVL sections obtained from normal controls and patients undergoing ureteral re-implantation for radiographically demonstrated PVUR. (A) Immunohistochemistry. Left to right: normal (nonrefluxing ureter) negative control, not stained with rabbit anti-TNXB antibody. There is absence of staining in all layers of the urothelial lining. Second panel: Normal control (nonrefluxing) ureter sample, stained with rabbit anti-TNX antibody (1:50). Note positive (brown) and equal (same intensity) staining throughout all of the urothelium. The third panel represents a refluxing vesicoureteral section of ureter, stained with rabbit anti-TNXB antibody (1:50). Note positive staining of all urothelial layers, but with a stronger intensity staining of the basal layer, compared with the normal control sample in the second panel. (B) Immunofluorescence. Left to right: normal (nonrefluxing ureter) negative control, not stained with rabbit anti-TNXB antibody. There is absence of staining in all layers of the urothelial lining, with nonspecific occasional superficial tissue auto fluorescence. Second panel: Normal control (nonrefluxing) ureter sample, stained with rabbit anti-TNXB antibody (1:50). Note positive (red fluorescence) and equal (same intensity) staining throughout all urothelial lining cell layers. The third panel represents a refluxing vesicoureteral section of ureter, stained with rabbit anti-TNX antibody (1:50). Note positive staining of all urothelial layers, which is brighter in the basal layer compared with the normal control sample in the second panel. Scale bar, μm 200.