Berns H et al. (2025), A homozygous human WNT11 variant is associated ...

XB-ART-61343

Dis Model Mech 2025 Apr 09; doi: 10.1242/dmm.052211.

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A homozygous human WNT11 variant is associated with laterality, heart and renal defects.

Berns H , Weber D , Haas M , Bakey Z , Brislinger-Engelhardt MM , Schmidts M , Walentek P .

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Wnt signaling plays important roles during vertebrate development, including left-right axis specification as well as heart and kidney organogenesis. We identified a homozygous human WNT11 variant in an infant with Situs inversus totalis, complex heart defects and renal hypodysplasia, and we used Xenopus embryos to functionally characterize this variant. WNT11c.814delG encodes a protein with reduced stability that lost signaling activity in vivo. This is remarkable, because the variant encodes a truncated ligand with nearly identical length and predicted structure to dominant-negative Wnts. Furthermore, we demonstrate that alteration of the truncated C-terminal end can restore stability and signaling activity similar to Xenopus dominant-negative Wnt11b. Our study also suggests similar functions for WNT11 in human development as described in model organisms. Therefore, biallelic WNT11 dysfunction should be considered as novel genetic cause in syndromal human phenotypes presenting with congenital heart defects and renal hypoplasia, with or without laterality defects. The work presented here enhances our understanding of human development and structure-function relationships in Wnt ligands.

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Species referenced: Xenopus laevis
Genes referenced: nodal1 pitx2 wnt11 wnt11b wnt3a wnt5a
GO keywords: Wnt signaling pathway

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	Fig. 1. A WNT11 variant associated with laterality, heart and kidney developmental defects (A) Pedigree indicating clinical features and segregation of the identified WNT11c.814delG variant amongst the three family members. (B) Chromatogram traces of Sanger sequencing results regarding the WNT11 variant segregation within the family, showing a homozygous deletion of c.814G in the affected child while both parents areheterozygous. In (A,B), I.1 = father; I.2 = mother; II.1 = son. (C,D) Alphafold 2.1.2 protein structure predictions of human (WNT11) and Xenopus (Wnt11b) Wnt ligands, and comparison of the WNT11c.814delG variant to dominant-negative (dn)Wnt11b. (D) Magnification of C-termini of truncated ligands with annotation of the last amino acids. Red arrows indicate C-terminal end.
	Fig. 1. A WNT11 variant associated with laterality, heart and kidney developmental defects (A) Pedigree indicating clinical features and segregation of the identified WNT11c.814delG variant amongst the three family members. (B) Chromatogram traces of Sanger sequencing results regarding the WNT11 variant segregation within the family, showing a homozygous deletion of c.814G in the affected child while both parents areheterozygous. In (A,B), I.1 = father; I.2 = mother; II.1 = son. (C,D) Alphafold 2.1.2 protein structure predictions of human (WNT11) and Xenopus (Wnt11b) Wnt ligands, and comparison of the WNT11c.814delG variant to dominant-negative (dn)Wnt11b. (D) Magnification of C-termini of truncated ligands with annotation of the last amino acids. Red arrows indicate C-terminal end.
	Fig. 2. Functional testing of the WNT11c.814delG variant in Xenopus embryos (A-D) Overexpression of mRNAs (at 70ng/l) encoding WNT11, Wnt11b, WNT11c.814delG and dnWnt11b, and analysis of effects on (A,B) morphogenesis at st. 13 (posterior view) and 20 (dorso-anterior view), (C,D) and left-right axis patterning (lateral views) by WMISH at st. 30 - 32 against pitx2c. (A,D) Representative images. In (A), neural folds (nf) and blastopores (bp) are indicated by yellow and magenta dashed lines, respectively. In (D), filled green arrowheads indicate normal left-sided expression, open white arrowheads indicate normal right-sided lack of expression. Filled magenta arrowheads indicate ectopic right-sided expression, open magenta arrowheads indicate loss of left-sided expression. (B,C) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. In (B), embryos were derived from 6 different batches (different parents) for Uninj. Ctrl., Inj. Ctrl., WNT11, WNT11c.814delG, and from 5 different batches for wnt11b and dnwnt11b conditions. In (C), embryos were derived from 4 different batches (different parents) for all conditions. In (A) and (D), all embryos from the same experiment are shown in the same magnification.
	Fig. 2. Functional testing of the WNT11c.814delG variant in Xenopus embryos (A-D) Overexpression of mRNAs (at 70ng/l) encoding WNT11, Wnt11b, WNT11c.814delG and dnWnt11b, and analysis of effects on (A,B) morphogenesis at st. 13 (posterior view) and 20 (dorso-anterior view), (C,D) and left-right axis patterning (lateral views) by WMISH at st. 30 - 32 against pitx2c. (A,D) Representative images. In (A), neural folds (nf) and blastopores (bp) are indicated by yellow and magenta dashed lines, respectively. In (D), filled green arrowheads indicate normal left-sided expression, open white arrowheads indicate normal right-sided lack of expression. Filled magenta arrowheads indicate ectopic right-sided expression, open magenta arrowheads indicate loss of left-sided expression. (B,C) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. In (B), embryos were derived from 6 different batches (different parents) for Uninj. Ctrl., Inj. Ctrl., WNT11, WNT11c.814delG, and from 5 different batches for wnt11b and dnwnt11b conditions. In (C), embryos were derived from 4 different batches (different parents) for all conditions. In (A) and (D), all embryos from the same experiment are shown in the same magnification.
	Fig. 3. Changes in C-terminal amino acids alters WNT11c.814delG function (A) Schematic representation of constructs and their lengths. Human WNT11 constructs are shown in blue, Xenopus Wnt11b constructs are shown in green. Orange indicates changes in the sequence. (B-E) Overexpression of mRNAs (at 70ng/l) encoding WNT11, WNT11c.814delG , WNT11814-1062 , WNT11850-1062 , dnWnt11b and Wnt11b811- 1059 , and analysis of effects on (B,C) morphogenesis at st. 17 (dorso-anterior view), and (D,E) left-right axis patterning (lateral views) by WMISH at st. 30 - 32 against pitx2c. (B,E) Representative images. In (B), neural folds (nf) are indicated by yellow dashed lines. In (E), filled green arrowheads indicate normal left-sided expression, open white arrowheads indicate normal right-sided lack of expression. Filled magenta arrowheads indicate ectopic right-sided expression, open magenta arrowheads indicate loss of leftsided expression. (C,D) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. In (C), embryos were derived from 6 different batches (different parents) for Uninj. Ctrl., Inj. Ctrl., WNT11814-1062 , WNT11850-1062 , Wnt11b811-1059, from 5 different batches for dnwnt11b, and from 3 different batched for WNT11c.814delG conditions. In (D), embryos were derived from 3 different batches (different parents) for all conditions. In (B) and (E), all embryos from the same experiment are shown in the same magnification.
	Fig. 3. Changes in C-terminal amino acids alters WNT11c.814delG function (A) Schematic representation of constructs and their lengths. Human WNT11 constructs are shown in blue, Xenopus Wnt11b constructs are shown in green. Orange indicates changes in the sequence. (B-E) Overexpression of mRNAs (at 70ng/l) encoding WNT11, WNT11c.814delG , WNT11814-1062 , WNT11850-1062 , dnWnt11b and Wnt11b811- 1059 , and analysis of effects on (B,C) morphogenesis at st. 17 (dorso-anterior view), and (D,E) left-right axis patterning (lateral views) by WMISH at st. 30 - 32 against pitx2c. (B,E) Representative images. In (B), neural folds (nf) are indicated by yellow dashed lines. In (E), filled green arrowheads indicate normal left-sided expression, open white arrowheads indicate normal right-sided lack of expression. Filled magenta arrowheads indicate ectopic right-sided expression, open magenta arrowheads indicate loss of leftsided expression. (C,D) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. In (C), embryos were derived from 6 different batches (different parents) for Uninj. Ctrl., Inj. Ctrl., WNT11814-1062 , WNT11850-1062 , Wnt11b811-1059, from 5 different batches for dnwnt11b, and from 3 different batched for WNT11c.814delG conditions. In (D), embryos were derived from 3 different batches (different parents) for all conditions. In (B) and (E), all embryos from the same experiment are shown in the same magnification.
	Fig. 4. WNT11c.814delG encodes a loss-of-function variant (A,B) Unilateral, right-sided overexpression of mRNAs (at 100ng/l) encoding WNT11c.814delG , WNT11814-1062 and WNT11850-1062, and analysis of effects on Wnt/- catenin signaling reporter (pbinLEF::dGFP) activity (green). (A) Representative images. (B) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. (C,D) Overexpression of mRNAs (at 100ng/l) encoding WNT11, WNT11c.814delG , WNT11814-1062 , WNT11850-1062 and Wnt11b, and analysis of (C) protein expression levels as well as (D) dimerization of ligands using Western blotting. -WNT11 = anti-human WNT11 antibody; -Tub. = antialpha-Tubulin antibody (loading control). Semi-quantitative analysis of WNT11 protein levels (graphs) show fold change of band intensities (normalized by loading control) over uninjected control sample (uninj. ctrl.). Magenta = upper bands (dimers; 50-80 kDa), cyan = lower bands (monomers; 25-45 kDa). Full membranes are shown in supplemental figure S3C and D. In (A), embryos were derived from 2 different batches (different parents) for all conditions, and all embryos are shown in the same magnification.
	Fig. 4. WNT11c.814delG encodes a loss-of-function variant (A,B) Unilateral, right-sided overexpression of mRNAs (at 100ng/l) encoding WNT11c.814delG , WNT11814-1062 and WNT11850-1062, and analysis of effects on Wnt/- catenin signaling reporter (pbinLEF::dGFP) activity (green). (A) Representative images. (B) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. (C,D) Overexpression of mRNAs (at 100ng/l) encoding WNT11, WNT11c.814delG , WNT11814-1062 , WNT11850-1062 and Wnt11b, and analysis of (C) protein expression levels as well as (D) dimerization of ligands using Western blotting. -WNT11 = anti-human WNT11 antibody; -Tub. = antialpha-Tubulin antibody (loading control). Semi-quantitative analysis of WNT11 protein levels (graphs) show fold change of band intensities (normalized by loading control) over uninjected control sample (uninj. ctrl.). Magenta = upper bands (dimers; 50-80 kDa), cyan = lower bands (monomers; 25-45 kDa). Full membranes are shown in supplemental figure S3C and D. In (A), embryos were derived from 2 different batches (different parents) for all conditions, and all embryos are shown in the same magnification.
	Fig. 4. WNT11c.814delG encodes a loss-of-function variant (A,B) Unilateral, right-sided overexpression of mRNAs (at 100ng/l) encoding WNT11c.814delG , WNT11814-1062 and WNT11850-1062, and analysis of effects on Wnt/- catenin signaling reporter (pbinLEF::dGFP) activity (green). (A) Representative images. (B) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. (C,D) Overexpression of mRNAs (at 100ng/l) encoding WNT11, WNT11c.814delG , WNT11814-1062 , WNT11850-1062 and Wnt11b, and analysis of (C) protein expression levels as well as (D) dimerization of ligands using Western blotting. -WNT11 = anti-human WNT11 antibody; -Tub. = antialpha-Tubulin antibody (loading control). Semi-quantitative analysis of WNT11 protein levels (graphs) show fold change of band intensities (normalized by loading control) over uninjected control sample (uninj. ctrl.). Magenta = upper bands (dimers; 50-80 kDa), cyan = lower bands (monomers; 25-45 kDa). Full membranes are shown in supplemental figure S3C and D. In (A), embryos were derived from 2 different batches (different parents) for all conditions, and all embryos are shown in the same magnification.
	Fig. 4. WNT11c.814delG encodes a loss-of-function variant (A,B) Unilateral, right-sided overexpression of mRNAs (at 100ng/l) encoding WNT11c.814delG , WNT11814-1062 and WNT11850-1062, and analysis of effects on Wnt/- catenin signaling reporter (pbinLEF::dGFP) activity (green). (A) Representative images. (B) Quantification of results. n = number of embryos analyzed per condition.  2 -test, p > 0.05 = ns; p < 0.05 = ; p < 0.01 = ; p < 0.001 = **. (C,D) Overexpression of mRNAs (at 100ng/l) encoding WNT11, WNT11c.814delG , WNT11814-1062 , WNT11850-1062 and Wnt11b, and analysis of (C) protein expression levels as well as (D) dimerization of ligands using Western blotting. -WNT11 = anti-human WNT11 antibody; -Tub. = antialpha-Tubulin antibody (loading control). Semi-quantitative analysis of WNT11 protein levels (graphs) show fold change of band intensities (normalized by loading control) over uninjected control sample (uninj. ctrl.). Magenta = upper bands (dimers; 50-80 kDa), cyan = lower bands (monomers; 25-45 kDa). Full membranes are shown in supplemental figure S3C and D. In (A), embryos were derived from 2 different batches (different parents) for all conditions, and all embryos are shown in the same magnification.
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