WNT Signaling Perturbations Underlie the Genetic Heterogeneity of Robinow Syndrome

Abstract

Locus heterogeneity characterizes a variety of skeletal dysplasias often due to interacting or overlapping signaling pathways. Robinow syndrome is a skeletal disorder historically refractory to molecular diagnosis, potentially stemming from substantial genetic heterogeneity. All current known pathogenic variants reside in genes within the noncanonical Wnt signaling pathway including ROR2, WNT5A, and more recently, DVL1 and DVL3. However, ∼70% of autosomal-dominant Robinow syndrome cases remain molecularly unsolved. To investigate this missing heritability, we recruited 21 families with at least one family member clinically diagnosed with Robinow or Robinow-like phenotypes and performed genetic and genomic studies. In total, four families with variants in FZD2 were identified as well as three individuals from two families with biallelic variants in NXN that co-segregate with the phenotype. Importantly, both FZD2 and NXN are relevant protein partners in the WNT5A interactome, supporting their role in skeletal development. In addition to confirming that clustered -1 frameshifting variants in DVL1 and DVL3 are the main contributors to dominant Robinow syndrome, we also found likely pathogenic variants in candidate genes GPC4 and RAC3, both linked to the Wnt signaling pathway. These data support an initial hypothesis that Robinow syndrome results from perturbation of the Wnt/PCP pathway, suggest specific relevant domains of the proteins involved, and reveal key contributors in this signaling cascade during human embryonic development. Contrary to the view that non-allelic genetic heterogeneity hampers gene discovery, this study demonstrates the utility of rare disease genomic studies to parse gene function in human developmental pathways.

Keywords: Frizzled; dual molecular diagnosis; human embryonic development; skeletal dysplasia.

Figure 1

Location of Identified Variants in DVL1, DVL3, and WNT5A Resulting in Dominant Robinow Syndrome The variants in DVL1 and DVL3 are mostly small insertions or deletions, except for two splicing variants in DVL3; all of them are predicted to lead to −1 frameshifting. Black rectangles represent transcript segments identical to the reference DVL1 (A), DVL3 (B), or WNT5A (C) mRNAs. Red rectangles indicate the shared transcript regions affected by the −1 frameshift in the predicted protein structure in all subjects. Part of exon 14 (DVL1) or exons 14 and 15 (DVL3) transcript sequence is shown in detail. Previously described variants are displayed by blue circles, whereas variants identified in this study are displayed by red circles. Larger circles represent identical variants in unrelated individuals. For complete description of all variants, see Tables S1 and S2.

Figure 2

Location of Identified Variants in FZD2 Segregating with the Associated Phenotypic Features (A) Pedigrees of the four probands with Robinow syndrome features, carrying variants in FZD2. (B) Sanger sequencing traces for index case subjects, demonstrating the detected variants at the nucleotide level. Family of BAB8596, far right, contains two variants in FZD2: p.Trp377^∗ and p.Pro142Leu. The stop gain was inherited from an affected mother and the missense represents a variant of uncertain significance inherited from father. (C) Representation of the known functional domains of FZD2 (green and red rectangles). Location of protein-coding variants identified in our cohort: one stop gain and three recurrent variants all affecting glycine 434 located within the third intracellular loop (red dots). One additional variant (p.Trp548^∗) from the literature, and reported in association with omodysplasia, is also included. (D) Photographs of consenting subjects with FZD2 variants demonstrating shared facial characteristics consisting of a high, broad forehead, prominent eyes, broad and low nasal bridge, low-set ears, broad nasal tip, and anteverted nares.

Figure 3

Identified Biallelic Variants in NXN (A) Pedigrees and Sanger sequencing traces for the two families with variants in NXN; a homozygous stop gain in BAB8841 (family HOU3189) and a heterozygous 3-bp in-frame deletion in BAB9847 and BAB9844 (family HOU3634). (B) High-density arrays and breakpoint mapping showing the second mutant allele in BAB9847 and BAB9844. The deletion is mediated by two highly similar Alu elements in direct orientation that flank the first coding exon. (C) Facial pictures demonstrating shared facial features including high forehead, prominent eyes, broad and low nasal bridge, broad nasal tip, anteverted nares, and micrognathia.

Figure 4

Robinow Syndrome-Associated Genes in the Wnt/PCP Pathway Identified in Human Subjects (A) Molecular diagnosis pie chart from the cohort of 21 Robinow syndrome-affected individuals who had a combination of direct Sanger screening and whole-exome sequencing to identify the molecular cause of their disorder highlighting the contribution of DVL1, DVL3, and FZD2 variants to RS. (B and C) The establishment of planar cell polarity is vital for vertebrate development; in humans, variants affecting genes in that pathway are linked to skeletal defects, including Robinow syndrome. In vitro studies from several model organisms have demonstrated that ROR2 binds to WNT5A and acts as a co-receptor with FZD2. The downstream effect is routed by the dishevelled homologs, which are stabilized by NXN in a context-dependent manner. The downstream readouts, which ultimately involves cytoskeletal reorganization, are a combination of small GTPases including RAC to activate JNK signaling. Pathogenic variants in all of the aforementioned genes have been identified in individuals with Robinow syndrome, underscoring the notion that this disorder results from aberrant Wnt/PCP signaling, likely due to disturbance of the organized development of chondrocytes at the growth plate.