Tomographic Study of the Malformation Complex in Correlation With the Genotype in Patients With Robinow Syndrome: Review Article

We aimed to understand the etiology behind the abnormal craniofacial contour and other clinical presentations in a number of children with Robinow syndrome. Seven children with Robinow syndrome were enrolled in this study (autosomal recessive caused by homozygous mutations in the ROR2 gene on chromosome 9q22, and the autosomal dominant caused by heterozygous mutation in the WNT5A gene on chromosome 3p14). In the autosomal recessive (AR) group, the main clinical presentations were intellectual, disability, poor schooling achievement, episodes of headache/migraine, and poor fine motor coordinative skills, in addition to massive restrictions of the spine biomechanics causing effectively the development of kyposcoliosis and frequent bouts of respiratory infections. Three-dimensional reconstruction computed tomography scan revealed early closure of the metopic and the squamosal sutures of skull bones. Massive spinal malsegmentation and unsegmented spinal bar were noted in the AR group. In addition to severe mesomelia and camptodactyly, in the autosomal dominant (AD) group, no craniosynostosis but few Wormian bones and the spine showed limited malsegemetation, and no mesomelia or camptodactyly have been noted. We wish to stress that little information is available in the literature regarding the exact pathology of the cranial bones, axial, and appendicular malformations in correlation with the variable clinical presentations in patients with the 2 types of Robinow syndrome.

DVL (DVL1; MIM 601365, DVL3; MIM: 601368), and NXN (MIM 612895). 7 These genes encode for components of the Wnt signaling, which controls convergent extension: a polarized cell migration that narrows and extends the body axis during vertebrate gastrulation as well as limb development and skeletal morphogenesis and regulates cell polarity, motility, survival, proliferation, and differentiation. [8][9][10][11] Autosomal recessive Robinow syndrome is typically the more severe form of RS caused by biallelic loss-of-function mutations in ROR2, WNT5A, and NXN. 5,7,12,13 Heterozygous mutations in the ROR2 gene is also associated with a distinct syndrome, the autosomal dominant brachydactyly type B1 (BDB1, OMIM 113000); however, a small phenotypic overlap has to be noted. 14,15 Homozygous mutations in ROR2 and WNT5A were found to impair the WNT5A/ROR2 signal pathway resulting in skeletal abnormalities and other features characteristic of RS. 8,[16][17][18] RS phenotype has been discussed comprehensively in zebrafish, Xenopus, and mouse models. Knockout of Ror2 and Wnt5a exhibited phenotypes similar to those found in RS patients. 16,[19][20][21] Hypoplastic phalanges in Ror2 −/− embryos were also shown as compared with the wild type, reflecting the mild brachydactyly seen in some ARRS patients. 22 Wnt5a was also found to be necessary for kidney development in both mouse and zebrafish phenocopied to RS patients. 5,15,16,23,24 Moreover, the NXN gene encodes nucleoredoxin, a regulator of the Wnt/PCP pathway through binding with DVL. Nxn −/− mouse models showed abnormal differentiation of osteoblastic cells, craniofacial abnormalities with a shortened nose, and cleft palate partially recapitulating the RS subjects' phenotype. 7,25 On the other hand, the milder autosomal dominant RS has been found to be caused by heterozygous mutations in WNT5A, DVL1, and DVL3. DVL is a highly conserved central mediator of Wnt signaling including the noncanonical Wnt/PCP pathway via interaction with ROR2 or by binding to the cytoplasmic frizzled family members and transducing the Wnt signal to downstream effectors. 26,27 All reported mutations in DVL1 and DVL3 led to perturbation of Wnt pathway and showed similar features associated with ADRS. 7,[28][29][30] Mice and Xenopus models for Dvl paralogs showed short stature, axial skeleton defects, and craniofacial malformations. 31,32 Recently identified variants in FZD2 (a Wnt receptor) associated ADRS and autosomal-dominant omodysplasia, an RS-like phenotype (OMOD2; OMIM 16475). 7,32,33 Fzd2 −/− mice also displayed lethal phenotype compatible with RS patients phenotype. 34 Furthermore, mutations in the receptor-like tyrosine kinase gene (RYK, MIM 600524) might also cause RS, but no mutations yet were identified in RS-affected patients. Ryk is essential for Wnt5a/PCP regulation in multiple developmental processes. Ryk together with the Ror2 were found to regulate the Wnt5a (Wnt/PCP) signaling via interacting with Vangl2. 12,35 These findings were supported in Ryk-deficient mice that showed typical PCP defects similar to Wnt5aand Ror2-null mice. [35][36][37] Therefore, mutations in human RYK might cause RS and brachydactyly and provide new insight into the etiology of these diseases.
Collectively, the pathogenesis of RS because of mutations in the RS-associated genes appears to be a result of perturbation of Wnt/PCP signaling. Locus heterogeneity characterizes a variety of skeletal dysplasia due to interacting or overlapping signaling pathways. The contribution of distinct genes from the same pathway may explain locus heterogeneity in RS.

Material and Methods
Ethical approval to report this case series was obtained from the Ethics Committee of the Turner Scientific Research Institute, No.3/2016, Pediatric Orthopedic Institute n.a. H. Turner, Department of Foot and Ankle Surgery, Neuroorthopaedics and Systemic Disorders, Parkovaya, Pushkin Saint-Petersburg, Russia. Written informed consent was obtained from the patients' guardians for their anonymized information to be published in this article. This study was conducted based on clinical and radiographic phenotypic interpretations of a group of children and was carried out between January 1, 2009, and March 2018. All patients showed the classical abnormalities of RS. Nevertheless, we noticed different clinical presentations between patients with AD and AR types of RS. The study included 7 unrelated patients (4 girls and 3 boys) who have been diagnosed and assessed by the first author. All presented clinically with the clinical and the radiological phenotypic characterizations of RS. The genotype confirmed the diagnosis of Robinow, and we further subdivided our group of patients into the AR form (3 patients underwent genetic tests; 2 of them showed a loss-of-function mutation in the ROR2 gene encoding a putative WNT5A receptor) and the AD form (4 patients; 2 of them showed mutations of WNT5A encoding a protein in the WNT signaling pathway). Conventional radiographic study via skeletal survey evaluation showed unclarified differences between the ARRS and the ADRS groups. The incentive to drive us to perform comprehensive tomographic analysis in both patient groups is to further understand the diversity in their complaints and the reasons behind the unusual clinical presentations.

Group I: Autosomal Recessive Pattern of Inheritance (RRS) Caused by Homozygous
Mutations in the ROR2 Gene on Chromosome 9q22 1. Stature: Growth deficiency of −4SD. 2. Developmental history and intelligence: All manifested subsequent developmental retardation and borderline intelligence with poor schooling achievements. 3. In this group, broad spectrum of clinical presentations such as intellectual disability, persistent headache, and episodes of dizziness have been recorded. Craniofacial contour has been analyzed via a 3-dimensional (3D) reconstruction computed tomography (CT) scan. A 7-year-old girl with AR type of RS complained of poor schooling achievements and persistent headache originating from the back of the skull. Three-dimensional reconstruction CT scan showed early closure of the metopic suture (arrowhead), associated with profound bossing of the frontal area (more marked at the glabella) notable features. Note the normality of the coronal sutures and acute depression of the nasofrontal angle with dysplastic supraorbital ridge ( Figure 1a). Threedimensional reconstruction CT scan in the same girl with ARRS showed early closure of the squamosal  pronation and supination. No camptodactyly, though rectangular first metacarpals, multiple pseudoepiphyses, and clinodactyly, were noted ( Figure 6; refer to Tables 1 and 2).

Discussion
The human skull is composed of several bones that fuse together after birth. The bones of the skull can be divided into the viscerocranium and the neurocranium. The viscerocranium consists of the bones that make up the bones of the face and the pharyngeal arches. The neurocranium consists of the bones protecting the brain and sensory organs. 38 The calvarium of the skull consists primarily of large flat bones separated by bony sutures. Craniosynostosis is a condition characterized by premature fusion of cranial sutures. The sutures generally fuse at the end of the second year of life. Previous studies described the extent of complications that might ensue in correlation with early fusion of the cranial sutures. 39 Surgical correction of craniosynostosis has to be done early in the child's life. The management of abnormal craniofacial anatomy, specifically the correction of craniosynostosis, is recommended between 3 and 6 months of age. 40 In our group of patients with autosomal recessive type of Robinow syndrome, it seems extremely late to intervene surgically in order to repair the synostotic sutures. Vertebral anomalies occurring during the mesenchymal stage may be     morbid complications such as respiratory insufficiency and pulmonary and cardiac hypertension, which characterize thoracic insufficiency syndrome, which might be fatal. Thoracic insufficiency syndrome is the inability of the thorax to ensure normal breathing. This clinical condition can be linked to variable axial anatomical disruptions such as fused ribs, hemivertebrae, and congenital unsegmented bars. The only article that described the craniofacial and intraoral phenotype of Robinow forms was the study by Beiraghi et al. 43 They concluded that there were differences in the severity of the craniofacial and intraoral features between the autosomal dominant and the recessive forms of RS. But, nevertheless, their findings did not include distinctive study of the skull bones and its correlation with the intraoral anatomy.

Conclusion
Tomographic studies are useful tools used to detect the pathological mechanism and to further understand the reasons behind the frequent clinical complaints in children/adults with syndromic malformation complex. Congenital osseous disruptions can hardly get interpreted via conventional radiographs, simply because of the anatomical overlap of the malformed osteogenic structures. To differentiate between the 2 types of RS and to further interpret the reasons behind the clinical ailments and the frequent complaints in these children, we referred to 3D reconstruction CT scan. We believe that tomographic studies could replace the high cost of molecular and genetic tests in RS patients, and it could alleviate the burden for the patients and their families.
Previous reports emphasized solely on the clinical management of orofacial pathologies in patients with RS. Scrutinizing the literature revealed insufficient explanation regarding the etiology behind the abnormal craniofacial contour, axial and appendicular malformations, and the correlated symptomatology. Early closure of the metopic suture and anterior part of the sagittal and the squamosal sutures requires special attention and early surgical interventions to overcome the intellectual disability and to lessen the episodes of migraine in the ARRS type. Similar plans are required to lessen the complications of axial and appendicular osteogenic deformities in the ARRS type. The connection between the broad-spectrum clinical presentations in RS with existing anatomical malformation complex has never been described in the literature. Three-dimensional reconstruction CT scan is the modality of choice for the precise assessment of the malformed skeletal system.

Author Contributions
Ali Al Kaissi conducted the study, and analyzed and drafted the manuscript. Vladimir Kenis contributed in study design and analysis. Mohammad Shboul and Susanne Gerit Kircher contributed in genetic analysis and laboratory investigations. Franz Grill and Rudolf Ganger revised the manuscript and provided critical analysis. All authors read and reviewed the final version of the manuscript.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethics Approval
Ethical approval to report this case series was obtained from the Ethics Committee of the Turner Scientific Research Institute

Informed Consent
Written informed consent was obtained from the patients' guardians for their anonymized information to be published in this article.