Neurogenic Defects Occur in LRIG2-Associated Urinary Bladder Disease

Abstract

Introduction: Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in HPSE2, whereas other rare families carry variants in LRIG2.LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction.

Methods: We describe a new family with LRIG2-associated UFS and studied Lrig2 homozygous mutant mice with ex vivo physiological analyses.

Results: The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant (c.1939C>T; p.Arg647∗). Lrig2 mutant mice had enlarged bladders. Ex vivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes.

Conclusion: Putting this family in the context of all reported UT disease-associated LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.

Keywords: LRIG2; Ochoa; bladder; neurogenic; syndrome; urofacial.

Graphical abstract

Figure 1

Identification of a novel pathogenic variant in LRIG2 in a family affected by urofacial syndrome. (a) Pedigree of affected family showing the inheritance of the pathogenic variant. (b) and (c) Facial appearance of the affected child, before and during smiling. (d–f) Cystourethrogram sequential images as the bladder was filled via a suprapubic catheter, showing vesicoureteric reflux (blue arrow) and an anatomically patent urethra (blue arrow). (g) Location of all identified LRIG2 pathogenic variants associated with UT disease. The uppermost alternating light and dark purple row indicates positions of LRIG2 exons and base position. Below this, the protein is depicted with its key domains indicated in different colors. Missense variants are shown above the protein sequence, whereas nonsense and frameshift variants are shown below the protein. The variant described in this paper is shown in bold and is located in the part of the gene coding for an immunoglobulin-like domain. WT, wild-type.

Figure 2

WT and Lrig2 Mut bladders. (a) Representative bladders at autopsy. Note the Mut bladders were distended with urine. Scale bars are 2 mm. (b) Bladder-to-body weight ratio was higher in Mut versus WT males (WT n = 6, Mut n = 7, P = 0.0018), and also in Mut versus WT female littermates (WT n = 6, Mut n = 8, P < 0.0001). (c) Nuclei number per area in the detrusor was similar between WT and mutant males (WT n = 3, Mut n = 5, P = 0.905), and between WT and mutant females (WT n = 5, Mut n = 5, P = 0.2506). (d) Percentage area positive for TGFβ1 staining in the detrusor was not different between male WT and mutant mice (WT n = 3, Mut n = 5, P = 0.819). It was also similar between female WT and mutant (WT n = 5, Mut n = 5, P = 0.315). (e) Cell numbers positive for the macrophage marker F4/80 in the detrusor were similar between male WT and mutants (WT n = 3, Mut n = 5, P = 0.3569) and between female WT and mutants (WT n = 5, Mut n = 5, P = 0.140). (f) Coherence of collagen fibrils in the detrusor layer was higher in male Mut compared with WT (WT n = 3, Mut n = 5, p = 0.0204) but it was similar between female WT and mutants (WT n = 5, Mut n = 5, P = 0.7256). (g) The percentage area positive for collagen staining in the detrusor was similar between male WT and mutants (WT n = 3, Mut n = 5, P = 0.434) but was similar between female WT and mutants (WT n = 5, Mut n = 5, P = 0.168). In this and in subsequent figures, WT males are represented by black filled squares, Mut males by purple open squares, WT females by black filled circles, and Mut females with purple open circles. Results are expressed as mean±SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 are the significant differences between Mut and WT of the same sex. WT, wild-type.

Figure 3

Dysfunctional outflow physiology in Lrig2 mutant mice. (a) Amplitudes of contractions evoked by 50 mM KCl in male WT (n = 6) and Mut (n = 7) outflow tracts. (b) Outflow relaxations in response to cumulatively increased concentrations of SNP in WT (n = 6) and mutant (n = 6) male outflow tracts. Curves show best fits to the Hill equation with EC₅₀ = 0.20 μM (control) and 0.24 μM (Mut) and E_max = 81.7 % (control) and 80.6 % (Mut). (c) Representative traces of WT and mutant male outflows contracted with 5 μM PE then stimulated by EFS at the frequencies indicated. (d) Mean ± SEM relaxations evoked by EFS, plotted as a function of frequency in male WT (n = 5) and Mut (n = 7) outflows; ∗∗P < 0.01. (e) Amplitudes of contraction evoked by 50 mM KCl in female outflows (WT n = 6, Mut n = 8). (f) Female outflow relaxations in response to cumulatively increased concentrations of SNP in WT (n = 6) and mutant (n = 8) outflow tracts. Curves show best fits to the Hill equation with EC₅₀ = 0.91 μM (control) and 0.61 μM (Mut) and E_max = 43.7 % (control) and 66.5 % (Mut). (g) Representative traces of WT and mutant female outflows contracted with 10 nM AVP then stimulated by EFS at the frequencies indicated. (h) Mean responses to EFS as a function of frequency in female outflows (WT n = 6, Mut n = 8). Mutant contraction and relaxation were plotted separately. Responses of Mut outflows tested in the presence of tetrodotoxin (N = 4) are also shown. Results are expressed as mean ± SEM. ∗P = 0.02 Mut versus tetrodotoxin, ∗∗∗∗P < 0.0001 WT compared with Mut. WT, wild-type.

Figure 4

Dysfunctional bladder body physiology in Lrig2 mutant mice. (a) Amplitudes of contractions evoked by 50 mM KCl in bladder rings from WT (n = 6) and Mut (n = 7) male mice. (b) Contraction of bladder rings from WT (n = 5) and Mut (n = 7) male mice in response to cumulative application of 10 nM – 50 μM carbachol, plotted as a function of carbachol concentration. Curves are the best fits of the Hill equation with EC₅₀ = 6.37 μM and E_max = 1.15 mN/mg in WT mice compared with EC₅₀ = 1.97 μM and E_max = 2.16 mN/mg in Mut mice. ∗P = 0.04 comparing WT and Mut by 2-way analysis of variance with repeated measures (c) Representative traces of contraction evoked in male WT and Mut bladder rings in response to EFS at the frequencies indicated. (d) Mean amplitudes of contraction evoked by EFS in bladders from male WT (n = 5) and Mut (n = 7) mice plotted as a function of frequency; ∗∗P = 0.0075. (e) Amplitudes of contractions evoked by 50 mM KCl in bladder rings from WT (n = 7) and Mut (n = 8) female mice. (f) Contraction of bladder rings from WT (n = 7) and Mut (n = 8) female mice in response to cumulative application of 10 nM – 50 μM carbachol, plotted as a function of carbachol concentration. Curves are the best fits of the Hill equation with EC₅₀ = 2.13 μM and E_max = 2.07 mN/mg in WT mice compared with EC₅₀ = 2.26 μM and E_max = 0.99 mN/mg in Mut mice. ∗P = 0.02 comparing WT with Mut by 2-way ANOVA. (g) Representative traces of contraction evoked in female WT and Mut bladder rings in response to EFS at the frequencies indicated. (h) Mean amplitudes of contraction evoked by EFS in bladders from female WT (n = 5) and Mut (n = 7) mice plotted as a function of frequency. ∗∗∗P = 0.0006 comparing WT and Mut by 2-way ANOVA with repeated measures. Results are expressed as mean ± SEM. WT, wild-type.