Characterization of the Robinow syndrome skeletal phenotype, bone micro-architecture, and genotype–phenotype correlations with the osteosclerotic form

1 ∣. INTRODUCTION

Robinow syndrome (RS) is a genetically heterogeneous disorder with a growing list of associated genes including autosomal recessive (OMIM: 268310), autosomal dominant, and an X-linked form of the disorder. Originally described by Robinow et al. (1969), RS was most notably characterized by distinct facial features, mesomelic limb shortening, and hypoplastic genitalia. However, it was not until 2000 when the identification of the first causative gene was found for the autosomal recessive form in ROR2 (Afzal et al., 2000; van Bokhoven et al., 2000). Since then, several more genes have been discovered including causative variants for autosomal dominant RS found in WNT5A (DRS1, OMIM 180700) (Person et al., 2010), DVL1 (DRS2, OMIM: 616331) (Bunn et al., 2015; White et al., 2015), and DVL3 (DRS3, OMIM: 616894) (White et al., 2016).

Not coincidentally, all of these genes play a role in the evolutionarily conserved Wnt signaling pathway. This complex system involves both canonical and noncanonical pathways, essential for several cellular functions including cell proliferation and primary embryonic axis formation (Niehrs, 2012; Strutt, Warrington, & Strutt, 2011). In vertebrae embryogenesis, the Wnt pathway is necessary for convergent-extension movements and limb bud outgrowth and patterning (Mlodzik, 2002). White et al (2018) recently proposed that RS is the result of perturbations of the Wnt pathway and in reviewing the previously estimated 70–80% of RS patients without a molecular diagnosis, identified new DRS causing genes, for example, FZD2, RRS, for example, NXN (RRS2, OMIM: 618529) and a potential X-linked form, for example, represented by the gene candidate GPC4.

Clinically, common features observed in both the dominant and recessive forms of RS include short stature, shortened extremities, brachydactyly, clinodactyly, and dysmorphic facies characterized by a prominent forehead, hypertelorism, large nasal bridge, anteverted nares, and midface hypoplasia. However, phenotypic differences exist with recessive ROR2-RS being classically associated with hemivertebrae, rib fusions, and scoliosis, while umbilical hernias, and supernumerary teeth are almost exclusively seen in patients with dominant RS (Mazzeu et al., 2007).

Considerable variation in the skeletal phenotype exist as well, including an osteosclerotic form of RS that has only been reported non-collectively in nine individuals in the current literature (Bunn et al., 2014, 2015; Eijkenboom, Verbist, Cremers, & Kunst, 2012; Kelly, Benson, Temtamy, Plotnick, & Levin, 1975; Shprintzen, Goldberg, Saenger, & Sidoti, 1982; White et al., 2015). Among these reports, some had isolated cranial osteosclerosis while others were more generalized, and in some cases the authors suggest that osteosclerosis may progress overtime (Bunn et al., 2014; Shprintzen et al., 1982). Secondary comorbidities have been described as well including conductive hearing loss due to appositional bone growth and narrowing of the middle ear (Eijkenboom et al., 2012). Notably, of the seven recently reported subjects, six had molecular confirmation of a DVL1 variant (Bunn et al., 2015; White et al., 2015), while the remaining was suspected based on phenotype and after WNT5A and ROR2 sequencing was normal (Eijkenboom et al., 2012). However, with so few cases to report, full characterization of the skeletal phenotype including the micro-architecture of the bones has yet to be explored. Therefore, we endeavored to better characterize the RS skeletal phenotype with a focus on the bone mineral content and density in a genetically diverse cohort of RS subjects utilizing dual-energy X-ray absorptiometry (DEXA) and high resolution peripheral quantitative computed tomography (HR-pQCT) scanning.

2 ∣. SUBJECTS AND METHODS

3 ∣. RESULTS

All of the seven unrelated subjects who participated in the study are male. This included four pediatric (<18 years) and three adult (≥18 years) subjects with an age range of 8–51 years (mean 19.6 years, SD 13.8). Molecularly, this cohort included four subjects with pathogenic variants in DVL1 as well as single individuals with pathogenic variants in WNT5A, ROR2, and GPC4 (Table 1). Of note, all of the participants had appeared in prior publications (Mazzeu et al., 2007; White et al., 2015, 2016, 2018; Zhang et al., 2020), although new data is presented in this study.

Physical examination with a focus on the musculoskeletal system revealed variability in limb shortening anomalies including mesomelia (4/7), rhizomelia (2/7), and micromelia (1/7). Additional common RS characteristic features seen in our cohort included brachydactyly (7/7), clinodactyly (7/7), camptodactyly (2/6), limited supination (3/7), genu valgus (1/7), scoliosis (1/7), and hemivertebrae (2/7) (Table 1).

DEXA scan (Figure 1) measures of BMC compared to height-matched controls revealed increased total body BMC (5/7), cranial BMC (7/7), and non-cranial BMC (3/7). DEXA scan measures of BMD compared to age-matched controls revealed increased total body BMD (3/7), cranial BMD (5/7), and non-cranial BMD (1/7) (Figure 2). Cranial osteosclerosis based on BMD for age was present in five of the seven subjects, one of which also had non-cranial osteosclerosis (Table 2).

Of those with DVL1-RS, when compared to height-matched controls, increased total BMC (3/4), cranial BMC (4/4), and non-cranial BMC (2/4) was observed. When compared to age-matched controls, increased total BMD (2/4), cranial BMD (4/4), and non-cranial BMD (1/4) was observed. The WNT5A-RS subject presented within two standard deviations of the mean for all of these measures except having an increased cranial BMC for height and low non-cranial BMD for age. The ROR2-RS subject had increased total, cranial, and non-cranial BMC for height, but normal BMD for all three measures. The GPC4-RS subject when compared to height matched controls had increased total and cranial BMC, but normal non-cranial BMC. Similarly, when compared to age-matched controls, this subject had increased total and cranial BMD but normal non-cranial BMD (Table 2).

Overall, cranial osteosclerosis was present in 5/7 subjects, 1 of which also had non-cranial osteosclerosis. Among this group was all 4 DVL1-RS subjects and the single GPC4-RS subject. In contrast, neither of the other RS patients with variants in WNT5A or ROR2 had evidence of osteosclerosis on their DEXA scans. Rather, mild osteopenia of the non-cranial skeleton was identified in the patient with WNT5A- RS, which may in part be related to the subject's history of malnutrition during infancy (Table 2).

The bone micro-architecture based on HR-pQCT imaging of the radius (Figure 3) was performed on six of the seven subjects, excluding the GPC4-RS subject. None of the subjects reported a history of the radial fractures or metal implants, so scans were performed on the left radius by default. Normative HR-pQCT data exist only for adult patients (Burt, Liang, Sajobi, Hanley, & Boyd, 2016; Zhu et al., 2018), of which three of the six subjects could reliably be interpreted (two with DVL1-RS and one with ROR2-RS). In general, all three of these adult subjects had lower cortical BMD of the radius, well below the 3%ile. The trabecular BMD, however, was slightly elevated in particular for the two adult DVL1-RS subjects, although still within two standard deviations. In contrast, the ROR2-RS subject had borderline low trabecular BMD. The number of trabeculations was also low for most including one DVL1-RS subject and one ROR2-RS subject among the three adults. However, the thickness of the trabeculations was well above the 97%ile for the DVL1-RS subjects, but within the normal range for the ROR2-RS subject (Figure 4).

4 ∣. DISCUSSION

Many of the major skeletal anomalies observed in RS such as shortened long bone, vertebral defects, and rib anomalies are easily identified on physical exam or X-ray imaging, and have been well described in the current literature. However, the complete scope of anomalies and particularly the micro-architecture of the bones in the osteosclerotic form of RS had not been formally assessed prior to this study. In this study, we endeavored to characterize the full spectrum of variability of microscopic bone structure anomalies based on BMC and BMD obtained through DEXA and HR-pQCT scanning. Additionally, we sought to determine whether the presence of osteosclerosis (cranial and non-cranial), which has been reported in a few recent case reports, may be associated with a particular RS genotype.

In regard to clinical physical examination of the musculoskeletal system, RS is classically associated with mesomelic limb shortening predominantly affecting the upper limbs, as was observed in 4/7 of our patients (Mazzeu et al., 2007; Roifman et al., 2015). More recent studies suggest some degree of genotype–phenotype correlation with 6/6 patients with FZD2-associated RS having either micromelia or mesomelia (White et al., 2018), whereas 3/3 patients with DVL1-associated RS had no mesomelia (Bunn et al., 2015). Also consistent with prior reports, our findings of additional skeletal features of RS included brachydactyly, clinodactyly, limited supination, genu valgus, scoliosis, and hemivertebrae. However, a full skeletal survey with X-ray imaging was not performed on all of the subjects for the purpose of this study, therefore, we cannot rule out the presence of other skeletal defects like rib or vertebral anomalies.

Based on DEXA scan imaging, we were able to identify a characteristic pattern of structural anomalies of the bones. While the osteosclerotic form of RS had been briefly reported before, it had not been explored in much depth until Bunn et al (Bunn et al., 2014) performed a DEXA scan on two subjects found to have significantly increased BMD. Our findings suggest that this is may be a much more common feature of RS with varying degrees of severity. Interestingly, all four patients in this study with DVL1-RS and one with GPC4-RS were found to have cranial osteosclerosis, while the single patients with a ROR2-RS and WNT5A-RS had normal or low BMD and BMC. Notably, our data suggest a clear predilection for osteosclerosis of the cranium among those affected, based on significantly elevated cranial BMD compared to non-cranial BMD. In fact, among the five with evidence of osteosclerosis, four had BMD measures within two standard deviations of the mean when not accounting for the cranium. The remaining subject had much more extensive osteosclerosis with a BMD well above the 97%ile for cranial and non-cranial measures, indicating the presence of generalized osteosclerosis of the bones. Similarly, BMC was elevated in the DVL1-RS and GPC4-RS subjects, and more specifically the cranial BMC, which may simply be a reflection of the increased BMD given the direct relationship of the two measures.

As is the case with other conditions known to cause cranial osteosclerosis, this can result in certain comorbidities. In particular, bilateral mixed hearing loss had been reported in four subjects with the osteosclerotic form of RS before (Bunn et al., 2014; Eijkenboom et al., 2012; Kelly et al., 1975; White et al., 2015). One of these subjects had evidence of hyperostosis of the skull, appositional bone growth of the middle ear walls with narrowing of the internal auditory canals, and thickened malformed ossicles found on CT scan. In this study, the GPC4-RS subject with cranial osteosclerosis also presented with hearing loss along with evidence of opacification of the bilateral mastoid and middle ear cavities. However, this subject also had choanal atresia and cranial nerve III, VI, and VII palsy secondary to foramen stenosis, which had not been reported in RS before. Thus, the severity of the osteosclerosis can cause considerable disability in patients with RS.

The mechanism by which osteosclerosis occurs in RS warrants additional investigation with little known at this time. In general, the Wnt signaling pathway is known to be a key regulator in bone health, as evident by defects in other genes in the pathway, including SOST and LRP5 causing a cranial osteosclerotic phenotype in Sclerosteosis (OMIM 269500) and Van Buchem disease (OMIM 607636) (Manolagas, 2014). In both conditions, a lack of sclerostin expression by osteocytes, which is a Wnt signaling antagonist, has been identified as the pathophysiological mechanism (Balemans et al., 2001; Kamiya et al., 2008; Li et al., 2008). Whether a similar mechanism exist in RS as well remains to be determined. Specifically, the roles of DVL1 and GPC4, which have been associated with the osteosclerotic form of RS in this study, warrant further investigation.

Interestingly, HR-pQCT revealed a unique pattern of radial bone micro-architecture for the DVL1-RS subjects with the increased BMD primarily affecting the trabecular bone as opposed to the cortical. There were also fewer trabeculations, but those that were present were generally thicker. However, these interpretations are understandably limited based on the fact that normative curves are not available for pediatric patients.

Additional limitations on this study include the general rarity of RS and, therefore, a low number of subjects available to report. Furthermore, our cohort only included single individuals with ROR2-RS and WNT5A-RS, which is insufficient to suggest that osteosclerosis could not occur with these forms. Interestingly, the single subject with GPC4-RS, however, did have cranial osteosclerosis, which was not an association made prior to this study.

In summary, we have presented a cohort of genetically diverse RS subjects to provide further evidence of a genotype–phenotype correlation, particularly relating to the presence of osteosclerosis. In this study, we have observed that osteosclerosis primarily affecting the skull appears to be a relatively common feature associated with DVL1-RS and GPC4-RS. Additionally, there is some variability within this group, with some patients being much more diffusely affected than others. The underlying mechanisms by which variants in DVL1 and GPC4 results in this phenotype remains unclear, but may have clinical management impact and require close monitoring as hearing loss and cranial nerve palsies can occur as a result of foramen stenosis. Therefore, patients with molecularly confirmed DVL1-RS and GPC4-RS and/or clinical evidence of osteosclerosis may benefit from routine hearing, vision, and cranial nerve evaluations. While DEXA or HR-pQCT have been used in this study to assess the effects of abnormal Wnt signaling in the bones of subjects with Robinow syndrome, the clinical utility of these imaging modalities is unproven as it is unclear at this point how it might guide clinical management. Nonetheless, much remains to be learned about this unique risk and patterns of osteosclerosis in RS and how it relates to the pathophysiology of the disorder.

DATA AVAILABILITY STATEMENT

The submission numbers for identified variants were deposited into ClinVar with the preliminary identifiers: SUB7033670. The dbGAP accession number for all exome sequences reported in this paper and for which informed consent for data sharing in controlled-access databases has been provided is dbGAP:phs000711.v5.p1.