Robinow syndrome is a rare developmental syndrome caused by variants in genes in Wnt signaling pathways. We previously showed that expression of patient variants in Dishevelled 1 (DVL1) in Drosophila and chicken models disrupts the balance of canonical and non-canonical Wnt signaling. In this study, we further examine morphological changes that occur due to expression of DVL11519ΔT, which serves as a prototype for other pathogenic variants. We show that epithelial imaginal disc development is disrupted in legs and wings and accompanied by increased cell death, without changes in cell proliferation. By inhibiting caspase-dependent cell death, we show that the altered epithelial morphology is not solely due to variant-induced cell death. Furthermore, we find alterations of basement membrane components and modulators. Notably we find ectopic Mmp1 expression and tissue distortion, which is dependent on JNK signaling. We also find an abnormal abundance of Drosophila collagen IV (Viking) in pupal wing development. Due to the complex nature of appendage development, we also examined the Bone Morphogenetic Protein pathway and found elevated signaling activity via the transcriptional readout dad-lacZ. Through these studies, we have gained more insight into the developmental consequences of DVL1 variants implicated in autosomal dominant Robinow syndrome.

Due to abnormal prenatal ultrasound findings of femoral shortening and flattened facial profile, a G2P0 pregnant patient underwent an amniocentesis at 15 weeks of gestation for proband-only exome sequencing. Bioinformatic filtering for genes included on the laboratory's extended skeletal dysplasia panel identified a heterozygous, likely pathogenic, frameshift variant in DVL1 NM_001330311.2:c.1575_1582dup; (p.Pro528ArgfsTer149). Pathogenic variants in DVL1 are associated with autosomal dominant Robinow syndrome (ADRS), a genetic disorder characterized by skeletal dysplasia with genital and craniofacial abnormalities. Prenatal ultrasound in the third trimester noted shortened long bones (first percentile for gestational age), macrocephaly with frontal bossing, short and upturned nose with a wide nasal root, triangular mouth, low pedal arches concerning for rocker-bottom feet, and ambiguous genitalia. A postnatal exam by Medical Genetics confirmed the prenatal findings in addition to hypertelorism, brachydactyly with broad thumbs and halluces, clinodactyly of second fingers, rigid gums with a frontal frenulum, and a sacral dimple. This case describes a novel variant in DVL1 identified in a fetus with prenatal and postnatal phenotypic features consistent with ADRS. To our knowledge, this is the first reported case of a prenatal molecular diagnosis of the dominant form of Robinow syndrome and the third case to describe prenatal ultrasound findings associated with this diagnosis.

Frizzled 2 (FZD2) is a transmembrane Wnt receptor. We previously identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus dishevelled-binding sequence. To model this variant, we used zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate a mouse allelic series. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with patient phenotypes. We also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each allele exhibit a highly penetrant cleft palate phenotype, shortened limbs compared with wild type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical Wnt signaling. In utero treatment with IIIC3a (a DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.

We report a family with a spectrum of short stature, craniofacial dysmorphism, and digital anomalies in a father and 2 daughters, with the youngest (proband) displaying a severe phenotype. Clinically, autosomal dominant Robinow syndrome (ADRS) was diagnosed. Whole-exome sequencing identified a heterozygous pathogenic BMP2 variant in the father and his daughters. The phenotype of short stature, facial dysmorphism, and skeletal anomalies with or without cardiac anomalies related to BMP2 haploinsufficiency has some facial and digital resemblance to ADRS. Although this variant segregated in the affected members, it failed to explain the severe phenotype of the proband. A reanalysis of the girl's raw data confirmed 2 disorders: a de novo likely pathogenic DVL1 variant implicated in ADRS and the familial BMP2 variant. A close interplay of high-throughput sequencing and deep phenotyping unraveled the complexities of the blended phenotype in the proband. Autosomal dominant Robinow syndrome (ADRS) is characterized by skeletal findings (short stature, mesomelic limb shortening predominantly of the upper limbs, and brachydactyly), genital abnormalities (in males: micropenis / webbed penis, hypoplastic scrotum, cryptorchidism; in females: hypoplastic clitoris and labia majora), dysmorphic facial features (widely spaced and prominent eyes, frontal bossing, anteverted nares, midface retrusion), dental abnormalities (including malocclusion, crowding, hypodontia, late eruption of permanent teeth), bilobed tongue, and occasional prenatal macrocephaly that persists postnatally. Less common findings include renal anomalies, radial head dislocation, vertebral abnormalities such as hemivertebrae and scoliosis, nail dysplasia, cardiac defects, cleft lip/palate, and (rarely) cognitive delay. When present, cardiac defects are a major cause of morbidity and mortality. A variant of Robinow syndrome, associated with osteosclerosis and caused by a heterozygous pathogenic variant in DVL1, is characterized by normal stature, persistent macrocephaly, increased bone mineral density with skull osteosclerosis, and hearing loss, in addition to the typical features described above. The diagnosis of autosomal dominant Robinow syndrome is established in a proband with typical suggestive findings and/or by the identification of a heterozygous pathogenic variant in DVL1, DVL3, or WNT5A through molecular genetic testing. Treatment of manifestations: Corrective surgeries as needed for cryptorchidism, abnormal penile insertion / penoscrotal position, and cleft lip/palate. Hormone therapy may be helpful for males with micropenis. Orthodontic treatment is typically required. Surveillance: Measurement of head circumference regularly in infancy and throughout childhood. Developmental assessment every three months in infancy and every six months to one year thereafter, or more frequently as needed if cognitive delays are identified. Dental evaluation every six to 12 months or as recommended. Periodic hearing assessments in childhood. Regular cardiac and renal assessment as needed by respective specialists if abnormalities are identified. Evaluation of relatives at risk: Evaluation of the sibs of a proband in order to identify as early as possible those who would benefit from institution of treatment and surveillance. Pregnancy management: Pregnancy in affected women appears to be generally uncomplicated. For an affected fetus, cesarean section may be required for abnormal presentation and/or cephalopelvic disproportion. ADRS is inherited in an autosomal dominant manner. A proband may have the disorder as a result of either an inherited or de novo pathogenic variant. Each child of an individual with ADRS has a 50% chance of inheriting the pathogenic variant; however, the severity of the clinical manifestations cannot be predicted from the results of molecular genetic testing. Prenatal testing for a pregnancy at increased risk is possible if the DVL1, DVL3, or WNT5A pathogenic variant has been identified in an affected family member.

Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.