Rare Variants in BNC2 Are Implicated in Autosomal-Dominant Congenital Lower Urinary-Tract Obstruction

Abstract

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853^∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.

Keywords: BNC2; LUTO; basonuclin 2; cloacae; distal pronephric outlet obstruction; functional genetics; lower urinary tract obstruction; posterior urethral valve; pronephric development; zebrafish.

Figure 1

Exome Sequencing and Targeted Re-sequencing in Families with LUTO Phenotype Identifies Variants in BNC2 (A and B) Exon structures of BNC2 cDNA of the two affected transcripts, ENST00000418777 and ENST00000380672, and protein domain structures are depicted. Positions of mutated nucleotides are indicated by arrows, and respective chromatograms are shown below. Evolutionary conservation among ortholog proteins is shown for family 2, carrying the p.His888Arg missense variant. The mutated amino acid residue is indicated with arrowheads and a red box. C2H2 = zinc-finger domain of the Cys2His2 folding group; C. elegans = Caenorhabditis elegans and D. melanogaster = Drosophila melanogaster. (C) Pedigree with multiple affected individuals in the index family 1. Affection of individuals varies in severity: II-4—meatal stenosis, pollakisuria, and nycturia; III-2—urethral stenosis, surgically corrected, frequent UTI, pollakisuria, and nycturia; III-4—urethral stenosis, frequent UTIs in youth, pollakisuria, and nycturia; IV-2—prenatal diagnosis in 13^th week of gestation with megacystis, urethral stenosis, surgically corrected and reconstructed. Individuals II-2 and I-2 are highlighted with the abbreviations E1 and E2 for unavailable genetic and clinical evaluation and represent potential BNC2 carriers who did not participate in our study. Triangles denote miscarriages and are annotated with respective weeks of gestation. E1 = unavailable genetic evaluation; E2 = unavailable clinical evaluation; and Wk = week of gestation. (D) Ventral micturation cystourethrogram (MCUG) image of affected individual IV-2; family 1 shows severe bilateral VUR grade V. (E) Pedigree with multiple affected individuals of family 2. Carriers of the identified BNC2 variant present with varying severity: I-2—no history of UTI or high voiding frequency, normal renal ultrasound; II-1—pollakisuria, nycturia, and pathological MCUG; and II-1—posterior urethral valve. I-2 is highlighted with a dot and represents a healthy carrier of the identified BNC2 variant. (F) Oblique MCUG image of affected individual III-2; family 2 shows severe right VUR grade V.

Figure 2

Embryonic Expression of BNC2 in Human, Murine, and Zebrafish Larvae Urinary-Tract Tissue (A) H&E-stained transverse section through a 7-week human embryo at the level of the urogenital sinus (ugs): the hindgut (hg), mesonephric ducts (md), and ureters (u) are also indicated. H. sapiens = Homo sapiens. (B) Magnification of ugs, corresponding to the boxed region in (A), shows positive BCN2 immunoreactivity (brown) in the primitive epithelium (arrowheads) and adjacent mesenchyme (asterisk). (C) There is only a faint background signal in this adjacent section in which the primary antibody was omitted. (D) Schematic overview depicting the human embryonic anatomy of the cross-section in (A). A red line indicates a sectional plane. (E and F) ISH with a pan-Bnc2 probe on a sagittal section of a representative E13.5 (TS21) mouse embryo. (E) Bnc2 expression is visible (in blue) in the brain, in the mandibular region, and in a small patch at the dorsal side above the spinal cord (white arrowheads top to bottom). Specific expression is furthermore observed in the urogenital region (black arrowheads). (F) Magnification (square in E) of the same embryo. Cells that express Bnc2 are found in the gt above the phallic urethra (phur) and below the pelvic urethra (plur) (black arrowheads). M. musculus = Mus musculus; pbl = primitive bladder (G) Schematic overview depicting the embryonic mouse anatomy of the cross-section in E. (H) Whole-mount ISH with an anti bnc2 probe shows (in purple) the expression of bnc2 RNA at 33 hpf in the pronephric ducts in WT zfl. In the lateral view (top), the pronephric duct is located above the yolk extension (black arrowheads), and in the dorsal view (bottom), the pairwise anlage of pronephric ducts are positively labeled. Sense controls (ctrl) did not show a staining. D. rerio = Danio rerio. (I) Immunohistochemistry staining (in brown) against Bnc2 on a sagittal paraffin cross-section of WT zebrafish at 100 hpf indicates Bnc2-positive cells in the pronephric duct (black arrowheads) but not in the intestine (black asterisk). (J) Schematic overview of pseudo-colored cloacal region of a 4 dpf zfl for better orientation and identification (modified according to Pyati et al.). K = kidney (red); G = gut (green); and C = cloaca (yellow). Scale bars represent 80 μm (A), 20 μm (B–C), 2 mm (E), 600 μm (F), 500 μm (H), and 100 μm (I)

Figure 3

Depletion of Bnc2 Causes Pericardial Effusion, Hydrocephalus, Glomerular Cysts, and Distal Pronephric-Outlet Obstruction in Zebrafish (A) Immunoblot analysis shows a protein decrease in bnc2 MO-injected zfl for Bnc2-202 (130 kDa, C7DZJ6 according to UniProt ID) but not for Bnc2-201 (110 kDa, F1R42 according to UniProt ID) at 2 days dpf, which is as predicted for the specific MO target side. UI = uninjected. (B) H&E staining of a sagittal section of a MO-injected zfl (lateral view, head to the left) shows (PE), hydrocephalus (HY), abnormal body curvature and a “vesicle” due to an outlet obstruction of the pronephric ducts (arrowhead in enlargement in B) at 56 hpf. (C) Zfl injected with bnc2 MO frequently develop a distal-outlet obstruction at 33 hpf; this obstruction is highlighted by an arrowhead in the enlargement of (C). (D) Whole-mount ISH with a pax2a probe relates the constituent parts of the bnc2 MO-induced pronephric-outlet obstruction to distal parts of the pronephric ducts and the cloacal region (black arrow). (E) ISH with a bnc2 probe in bnc2 MO-injected embryos shows bnc2 mRNA expression in relevant tissues forming a pronephric-outlet obstruction (black arrow). (F and G) Zfl injected with bnc2 MO develop glomerular cysts (white arrowheads in G) and dilatation of the pronephric ducts (white asterisk in G). Images from in vivo observation through fluorescence microscopy (dorsal view) in Tg(wt1b:GFP) were taken at 54 hpf in zfl injected with control (Ctrl) MO (F) and bnc2 MO (G). Tg = transgenic zebrafish line. (H) The graph shows 100% of all at 1 dpf surviving zfl of the five different cohorts: UI, Ctrl MO, bnc2 MO, bnc2 MO + WT human RNA, and bnc2 MO + mutated human RNA (transcript ENST00000380672) bearing the p.His888Arg variant (for absolute numbers, see Figure 3H). A distal “vesicle” due to an outlet obstruction (phenotype) of the pronephric ducts can be seen in 21% of bnc2 MO-injected zfl compared to 0% of zfl with a “vesicle” in both control groups (p < 0.05, unpaired t test). 6% of zfl injected with bnc2 MO + WT mRNA develop a “vesicle,” and so do 16% of zfl injected with bnc2 MO + p.H888R. Data are presented as means with standard error of the mean (SEM). (I) Quantification of glomerular cyst rates (phenotype) in Tg(wt1b:GFP) zfl at 2 dpf depicts significantly (p < 0.05, unpaired t test) clear glomerular cysts in 57% of bnc2 MO-injected zfl. 26% of zfl injected with bnc2 MO + WT RNA develop pronephric cysts; 51% of those injected with bnc2 MO + p.His888Arg RNA develop pronephric cysts. Data are presented as means with SEM. (J) Quantification of death rates at up to 3 dpf shows an increased mortality up to 64% in bnc2 MO-injected zfl compared to zfl injected with UI (7%) and Ctrl MO (6%). With bnc2 MO injected, zfl show significantly (p < 0.0001, Mantel-Cox test) reduced survival. Co-injection of bnc2 MO + human WT RNA results in a mortality of 69%. Aggravated mortality up to 81% can be detected in the zfl injected with bnc2 MO + p.His888Arg RNA. Scale bars represent 200 μm (B), 50 μm (magnification in B), 500 μm (C), 100 μm (magnification in C), and 100 μm (D–G). ^∗∗p < 0.005, ^∗∗∗ p < 0.0005.

Figure 4

Distal Pronephric-Outlet Obstruction Is Formed by Pronephric Tissue and Causes Dilated Pronephric Ducts and Increased Apoptosis (A) Tg(HGj4A) GFP-reporter zfl (lateral view) injected with bnc2 MO emphasize the belonging of the distal pronephric-outlet obstruction, visible as a “vesicle” (marked with a white asterisk), to the distal pronephric duct and cloaca. The enlarged inlay shows the same “vesicle” in the sole bright-field image. (B and C) High-resolution two-photon microscopy dorsal view of the cloacal region (white asterisks) in Tg(HGj4A) zfl injected with Ctrl MO (B) and bnc2 MO (C) indicates the intimate relation of the pronephric-outlet obstruction to the bilateral pronephric ducts. Widths of the pronephric-duct lumen are indicated with white arrows. (D) Quantification of pronephric-duct widths in Tg(HGj4A) zfl injected with bnc2 MO shows significantly (p = 0.0002, Student’s t test) dilated pronephric ducts compared to controls. PD = pronephric ducts. Data are presented as means with SEM. (E–G) Cleaved caspase 3 staining in bnc2 MO-injected zfl (56 hpf) shows an increased rate of apoptosis (black arrowheads in G) compared to Ctrl MO-injected embryos (E–F) around the pronephric duct (marked by red lines), the cloacal region, and the central nervous system (not shown). An overview emphasizing and localizing the pronephric regions shown in (F) and (G) is shown in €. (H and I) Dorsal image from immunofluorescence staining of acetylated tubulin-stained cilia (red, acetTub) and GFP (green) depicts normal cilia morphology in the pronephric ducts in Tg(wt1b:GFP) zfl injected with Ctrl MO (H) and bnc2 MO (I) at 56 hpf. Pronephric ducts are widened in the bnc2 MO zfl as mentioned before. White arrowheads point to glomerular cysts in the bnc2 MO zfl. The sole acetylated-tubulin stain (red channel) emphasizes cilia morphology in the respective enlargement inlays of (H) and (I). Scale bars represent 200 μm (A), 50 μm (magnification of A–C), 100 μm (E–G), 50 μm (H and I), and 25 μm (magnifications of H and I). ^∗∗∗ p < 0.0005.