Novel pathogenic variants and quantitative phenotypic analyses of Robinow syndrome: WNT signaling perturbation and phenotypic variability

Abstract

Robinow syndrome (RS) is a genetically heterogeneous disorder with six genes that converge on the WNT/planar cell polarity (PCP) signaling pathway implicated (DVL1, DVL3, FZD2, NXN, ROR2, and WNT5A). RS is characterized by skeletal dysplasia and distinctive facial and physical characteristics. To further explore the genetic heterogeneity, paralog contribution, and phenotypic variability of RS, we investigated a cohort of 22 individuals clinically diagnosed with RS from 18 unrelated families. Pathogenic or likely pathogenic variants in genes associated with RS or RS phenocopies were identified in all 22 individuals, including the first variant to be reported in DVL2. We retrospectively collected medical records of 16 individuals from this cohort and extracted clinical descriptions from 52 previously published cases. We performed Human Phenotype Ontology (HPO) based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. Individuals with FZD2 variants clustered into two groups with demonstrable phenotypic differences between those with missense and truncating alleles. Probands with biallelic NXN variants clustered together with the majority of probands carrying DVL1, DVL2, and DVL3 variants, demonstrating no phenotypic distinction between the NXN-autosomal recessive and dominant forms of RS. While phenotypically similar diseases on the RS differential matched through HPO analysis, clustering using phenotype similarity score placed RS-associated phenotypes in a unique cluster containing WNT5A, FZD2, and ROR2 apart from non-RS-associated paralogs. Through human phenotype analyses of this RS cohort and OMIM clinical synopses of Mendelian disease, this study begins to tease apart specific biologic roles for non-canonical WNT-pathway proteins.

Keywords: DVL2; HPO terms; genotype-phenotype correlation; indel mutations; molecular diagnosis; quantitative phenotyping cluster heatmap; screw-tail breed dogs; skeletal dysplasia; traits and OMIM clinical synopsis.

Figure 1

Map location of identified variants in RS-related genes resulting in AD-RS Previously described variants (blue circles) and variants identified in this study (red circles) are shown. Larger circles represent identical variants in unrelated individuals; number of recurrent variants is proportional to size of circle. Chromosome and cytogenetic interval location of the canonical transcripts are provided: individual exons (black rectangles) on a horizontal line are drawn to represent gene structure. (A) Variants within the coding region of DVL1 resulting in AD-RS: variants are mostly small insertions or deletions, except for one splicing variant; all of them are predicted to lead to −1 frameshifting of the reading frame. Zoomed-in view shows the bases affected by variants in exon 14 (i.e., penultimate exon). PTC created by −1 frameshifting variants is displayed by an orange diamond. (B) Variant within the coding region of DVL2 resulting in AD-RS: BAB14964 was found to have a de novo 1-bp duplication at the position c.2105dupC; p.Pro703Serfs∗103. (C) Variants within the coding region of DVL3 resulting in AD-RS: the variants in DVL3 are mostly small insertions or deletions, except for four splicing variants. All mutations, analogous to DVL1, are predicted to lead to −1 frameshifting of the reading frame. PTC created by −1 frameshifting variants is displayed by an orange diamond. Zoomed-in view shows the bases affected by variants in exon 14 and 15. (D) Predicted protein translational effects of FZD2 variants resulting in AD-RS: pathogenic variants in FZD2 are missense or truncating variants affecting mostly the Frizzled domain. (E) Predicted protein translational effects of WNT5A variants resulting in AD-RS: pathogenic or likely pathogenic variants in WNT5A are mostly missense, except for two indels. The majority of WNT5A variants involve substitution or creation of a cysteine residue.

Figure 2

Phenotypic analysis of subjects with pathogenic variants in DVL1, DVL3, FZD2, WNT5A, and NXN Prevalence (0–1.0) of phenotypes in subjects with pathogenic variants in DVL1, DVL3, FZD2, WNT5A, and NXN shown in Table S2 is displayed by heatmap. Within the heatmap, black indicates a higher prevalence, while light peach color indicates lower prevalence; light gray indicates these specific data are not available. The phenotypes are listed in order of overall decreasing prevalence. A key is provided on the right.

Figure 3

RS cohort summary and genotype-phenotype characterization (A) Cohort description. Clinical notes and molecular diagnoses of 68 subjects were collected, including 16 subjects in this study and 52 from previously published subjects. (B) HAC and visualization of quantitative phenotypic similarity allow refinement of genotype-phenotype correlations in RS. The dendrogram shown at the top and to the left of the heatmap is based on HAC analysis of the dissimilarity matrix produced from Lin semantic similarity scores and with k set to 6. Unique clusters are represented by different colors, and individual probands are labeled on top of and to the right of the heatmap. Within the heatmap, dark red indicates a higher similarity, while dark blue indicates lower similarity. A key is provided on the left. Blue rectangle: 84% of DVL1 and DVL3 individuals as well as DVL2 subject (highlighted by a red dashed rectangle) grouped into these two clusters in addition to the NXN individuals. Green rectangle: 62.5% (N = 5 out of 8) of FZD2 individuals grouped in this cluster have missense variants. Purple rectangle: 100% (N = 5 out of 5) of FZD2 individuals grouped in this cluster have truncating variants. Black rectangle: FZD2 outlier subject (FZD2_Subject31), whose clinical phenotypes are less similar to RS individuals. Black dashed rectangle: NXN_Subject58, who has a ∼1-Mb deletion. (C) Variants observed in NXN resulting in AR-RS: variants are displayed by blue circles; larger circles represent identical variants in unrelated individuals. The NXN subject marked as black dashed rectangle harbors a nonsense variant (SNV) in trans with an ∼1-Mb telomeric deletion (CNV), whereas the other three individuals all have biallelic LoF variants affecting NXN. Two of them from the same family were found to have compound heterozygous variants of an in-frame deletion and an Alu-Alu-mediated exonic deletion of the entire exon 1. (D) Patients with variants affecting FZD2 are separated in two distinct clusters based on phenotypic similarity score. Most of the individuals with missense variants affecting Gly434 in FZD2 were grouped into the missense cluster (green rectangle), whereas most of the individuals with truncating variants at C-terminal end of FZD2 were grouped into the truncating cluster (purple rectangle). Variants are displayed (blue circles) with larger circles representing identical variants in unrelated individuals. One outlier individual (FZD2_Subject31, black rectangle) was identified to have a truncating variant affecting the N terminus of the Frizzled-like domain.

Figure 4

Quantitative similarity analysis of RS phenotype supports high similarity of known differential diagnoses and indicate lack of similarity with diseases caused by variants affecting paralogous WNT-pathway genes (A) Gap statistic curve for heatmap displayed in (D). Gap statistic for RS, RS-like, WNT paralog-associated diseases, and top disease-associated gene phenotype matches (p < 0.001) is displayed on the y axis and number of clusters tested on the x axis. The point on the curve where slope changed from a trend of higher to lower (i.e., additional numbers of clusters were not adding as much to the gap statistic) was used to choose optimal number of clusters (k = 7). (B) Gap statistic plot to determine number of clusters to display by heatmap in (E). Gap statistic for RS, RS-like, WNT paralog-associated diseases, and top disease phenotype matches (p < 0.001) is displayed on the y axis and number of clusters tested on the x axis. The gap statistic curve along with the heatmap representation in (C) were used to select the number of clusters that both represented the visually appreciable clusters in the heatmap and the greatest increase in gap statistic (k = 7). (C) Number of WNT, FZD, and ROR paralogs in human. (D and E) Semantic similarity heatmap results between disease-associated phenotypes of RS, RS phenocopies, WNT-associated disease phenotypes, and significantly similar known disease gene phenotypes (D) and OMIM-annotated disease phenotypes (E). Red rectangle: RS caused by DVL1, DVL3, WNT5A, ROR2, and NXN are clustered together with RS genes. Black arrow: the most frequent differential diagnosis of RS, Aarskog-Scott syndrome, has the highest similarity scores with RS. Green rectangle: most of the differential diagnoses observed in our in-house cohort are clustered together with RS and RS genes. Gray rectangle: diseases caused by WNTs, FZDs, or RORs not specific in WNT/PCP signaling, which can be distinguished from the RS cluster. Unique clusters are represented by different colors, and diseases/genes are labeled on top of and to the right of the heatmap. Within the heatmap, dark red indicates a higher similarity, while dark blue indicates lower similarity. A legend is provided on the left-hand side.

Figure 5

Photographs and radiographs of BAB14964 who has a likely pathogenic variant in DVL2 (A) Facial characteristics include a tall forehead with high anterior hairline, hypertelorism, low nasal bridge, low-set ears, smooth philtrum, downturned corners of the mouth, and micro-retrognathia; standing photographs show micromelia with mesomelic predominance and genu valgus. (B–F) Selected radiographs from skeletal survey at 2 days old. (B) Chest radiograph and (C) magnified view: there are 11 rib pairs, with small accessory rib ossicles bilaterally, arising between the seventh and eighth ribs (white arrows). A resolving sagittal cleft is also shown in the T6 vertebral body (black arrow). There is a possible anterior rib fusion anomaly of the left first and second ribs. The T9 and T10 vertebral bodies are mildly hypoplastic, more easily seen on the lateral spine radiograph (D). Radiographs of upper limb (E) and lower limb (F) show diffuse osteosclerosis with some narrowing of the medullary spaces. There is mild mesomelic shortening of the upper limb, and mild rhizomelic shortening of the lower limb. (G and H) Radiographs of left arm (G) and left hand (H) at age 3 years. Mesomelic shortening is now more pronounced but remains mild. Diffuse osteosclerosis is no longer apparent. There is hypoplasia of the first metacarpal, second and fifth distal phalanges, and fifth middle phalanx, with associated clinodactyly of the little finger.

Figure 6

Quantitative similarity analysis of subjects carrying pathogenic variants affecting FZD2 reveal phenotypic clustering with RS Phenotypic data from 16 subjects with pathogenic FZD2 variants were compiled and similarity analysis was performed against RS OMIM genes (n = 6) and other mesomelic/rhizo-mesomelic dysplasias (n = 9), including omodysplasia 1 and 2 as per Mortier et al. Left: gap statistic, a cutoff value of p < 0.001 for similarity was used to define the group of diseases used for gap statistic testing, which was subsequently used to choose the number of clusters (k = 2). Right: HAC and heatmap for mesomelic/rhizo-mesomelic dysplasias, FZD2 subjects, and significant disease matches.