An Additional Lrp4 High Bone Mass Mutation Mitigates the Sost-Knockout Phenotype in Mice by Increasing Bone Remodeling.

Pathogenic variants disrupting the binding between sclerostin (encoded by SOST) and its receptor LRP4 have previously been described to cause sclerosteosis, a rare high bone mass disorder. The sclerostin-LRP4 complex inhibits canonical WNT signaling, a key pathway regulating osteoblastic bone formation and a promising therapeutic target for common bone disorders, such as osteoporosis. In the current study, we crossed mice deficient for Sost (Sost-/-) with our p.Arg1170Gln Lrp4 knock-in (Lrp4KI/KI) mouse model to create double mutant Sost-/-;Lrp4KI/KI mice. We compared the phenotype of Sost-/- mice with that of Sost-/-;Lrp4KI/KI mice, to investigate a possible synergistic effect of the disease-causing p.Arg1170Trp variant in Lrp4 on Sost deficiency. Interestingly, presence of Lrp4KI alleles partially mitigated the Sost-/- phenotype. Cellular and dynamic histomorphometry did not reveal mechanistic insights into the observed phenotypic differences. We therefore determined the molecular effect of the Lrp4KI allele by performing bulk RNA sequencing on Lrp4KI/KI primary osteoblasts. Unexpectedly, mostly genes related to bone resorption or remodeling (Acp5, Rankl, Mmp9) were upregulated in Lrp4KI/KI primary osteoblasts. Verification of these markers in Lrp4KI/KI, Sost-/- and Sost-/-;Lrp4KI/KI mice revealed that sclerostin deficiency counteracts this Lrp4KI/KI effect in Sost-/-;Lrp4KI/KI mice. We therefore hypothesize that models with two inactivating Lrp4KI alleles rather activate bone remodeling, with a net gain in bone mass, whereas sclerostin deficiency has more robust anabolic effects on bone formation. Moreover, these effects of sclerostin and Lrp4 are stronger in female mice, contributing to a more severe phenotype than in males and more detectable phenotypic differences among different genotypes.

Heterozygous pathogenic variants involving CBFB cause a new skeletal disorder resembling cleidocranial dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (RUNX2) is currently the only known disease-causing gene for CCD, but several studies have suggested locus heterogeneity. METHODS: The cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level. RESULTS: In each subject a heterozygous pathogenic variant in CBFB was detected, whereas no genomic alteration involving RUNX2 was found. Three CBFB variants (one splice site alteration, one nonsense variant, one 2 bp duplication) were shown to result in a premature stop codon. A large intragenic deletion was found to delete exon 4, without affecting CBFB expression. The effect of a second splice site variant could not be determined but most likely results in a shortened or absent protein. Affected individuals showed similarities with RUNX2-related CCD, including dental and clavicular abnormalities. Normal stature and neurocognitive problems were however distinguishing features. CBFB encodes the core-binding factor ß subunit, which can interact with all RUNX proteins (RUNX1, RUNX2, RUNX3) to form heterodimeric transcription factors. This may explain the phenotypic differences between CBFB-related and RUNX2-related CCD. CONCLUSION: We confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants in CBFB in a cohort of eight individuals with clinical and radiographic features reminiscent of CCD.

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cß subunits of PKA during human development.

Delineation of a new fibrillin-2-opathy with evidence for a role of FBN2 in the pathogenesis of carpal tunnel syndrome.

BACKGROUND: Although carpal tunnel syndrome (CTS) is the most common form of peripheral entrapment neuropathy, its pathogenesis remains largely unknown. An estimated heritability index of 0.46 and an increased familial occurrence indicate that genetic factors must play a role in the pathogenesis. METHODS AND RESULTS: We report on a family in which CTS occurred in subsequent generations at an unusually young age. Additional clinical features included brachydactyly and short Achilles tendons resulting in toe walking in childhood. Using exome sequencing, we identified a heterozygous variant (c.5009T>G; p.Phe1670Cys) in the fibrillin-2 (FBN2) gene that co-segregated with the phenotype in the family. Functional assays showed that the missense variant impaired integrin-mediated cell adhesion and migration. Moreover, we observed an increased transforming growth factor-ß signalling and fibrosis in the carpal tissues of affected individuals. A variant burden test in a large cohort of patients with CTS revealed a significantly increased frequency of rare (6.7% vs 2.5%-3.4%, p<0.001) and high-impact (6.9% vs 2.7%, p<0.001) FBN2 variants in patient alleles compared with controls. CONCLUSION: The identification of a novel FBN2 variant (p.Phe1670Cys) in a unique family with early onset CTS, together with the observed increased frequency of rare and high-impact FBN2 variants in patients with sporadic CTS, strongly suggest a role of FBN2 in the pathogenesis of CTS.

Bi-allelic Loss-of-Function Mutations in the NPR-C Receptor Result in Enhanced Growth and Connective Tissue Abnormalities.

The natriuretic peptide signaling pathway has been implicated in many cellular processes, including endochondral ossification and bone growth. More precisely, different mutations in the NPR-B receptor and the CNP ligand have been identified in individuals with either short or tall stature. In this study we show that the NPR-C receptor (encoded by NPR3) is also important for the regulation of linear bone growth. We report four individuals, originating from three different families, with a phenotype characterized by tall stature, long digits, and extra epiphyses in the hands and feet. In addition, aortic dilatation was observed in two of these families. In each affected individual, we identified a bi-allelic loss-of-function mutation in NPR3. The missense mutations (c.442T>C [p.Ser148Pro] and c.1088A>T [p.Asp363Val]) resulted in intracellular retention of the NPR-C receptor and absent localization on the plasma membrane, whereas the nonsense mutation (c.1524delC [p.Tyr508∗]) resulted in nonsense-mediated mRNA decay. Biochemical analysis of plasma from two affected and unrelated individuals revealed a reduced NTproNP/NP ratio for all ligands and also high cGMP levels. These data strongly suggest a reduced clearance of natriuretic peptides by the defective NPR-C receptor and consequently increased activity of the NPR-A/B receptors. In conclusion, this study demonstrates that loss-of-function mutations in NPR3 result in increased NPR-A/B signaling activity and cause a phenotype marked by enhanced bone growth and cardiovascular abnormalities.

A Panel-Based Sequencing Analysis of Patients with Paget's Disease of Bone Suggests Enrichment of Rare Genetic Variation in regulators of NF-κB Signaling and Supports the Importance of the 7q33 Locus.

Paget's disease of bone (PDB) is a common bone disorder characterized by focal lesions caused by increased bone turnover. Monogenic forms of PDB and PDB-related phenotypes as well as genome-wide association studies strongly support the involvement of genetic variation in components of the NF-κB signaling pathway in the pathogenesis of PDB. In this study, we performed a panel-based mutation screening of 52 genes. Single variant association testing and a series of gene-based association tests were performed. The former revealed a novel association with NFKBIA and further supports an involvement of variation in NR4A1, VCP, TNFRSF11A, and NUP205. The latter indicated a trend for enrichment of rare genetic variation in GAB2 and PRKCI. Both single variant tests and gene-based tests highlighted two genes, NR4A1 and NUP205. In conclusion, our findings support the involvement of genetic variation in modulators of NF-κB signaling in PDB and confirm the association of previously associated genes with the pathogenesis of PDB.

Conditional mouse models support the role of SLC39A14 (ZIP14) in Hyperostosis Cranialis Interna and in bone homeostasis.

Hyperostosis Cranialis Interna (HCI) is a rare bone disorder characterized by progressive intracranial bone overgrowth at the skull. Here we identified by whole-exome sequencing a dominant mutation (L441R) in SLC39A14 (ZIP14). We show that L441R ZIP14 is no longer trafficked towards the plasma membrane and excessively accumulates intracellular zinc, resulting in hyper-activation of cAMP-CREB and NFAT signaling. Conditional knock-in mice overexpressing L438R Zip14 in osteoblasts have a severe skeletal phenotype marked by a drastic increase in cortical thickness due to an enhanced endosteal bone formation, resembling the underlying pathology in HCI patients. Remarkably, L438R Zip14 also generates an osteoporotic trabecular bone phenotype. The effects of osteoblastic overexpression of L438R Zip14 therefore mimic the disparate actions of estrogen on cortical and trabecular bone through osteoblasts. Collectively, we reveal ZIP14 as a novel regulator of bone homeostasis, and that manipulating ZIP14 might be a therapeutic strategy for bone diseases.

WNT16 Requires Gα Subunits as Intracellular Partners for Both Its Canonical and Non-Canonical WNT Signalling Activity in Osteoblasts.

In the past years, WNT16 became an interesting target in the field of skeletal research, as it was identified as an essential regulator of the cortical bone compartment, with the ability to increase both cortical and trabecular bone mass and strength in vivo. Even though there are indications that these advantageous effects are coming from canonical and non-canonical WNT-signalling activity, a clear model of WNT signalling by WNT16 is not yet depicted. We, therefore, investigated the modulation of canonical (WNT/ß-catenin) and non-canonical [WNT/calcium, WNT/planar cell polarity (PCP)] signalling in human embryonic kidney (HEK) 293 T and SaOS2 cells. Here, we demonstrated that WNT16 activates all WNT-signalling pathways in osteoblasts, whereas only WNT/calcium signalling was activated in HEK293T cells. In osteoblasts, we therefore, additionally investigated the role of Gα subunits as intracellular partners in WNT16's mechanism of action by performing knockdown of Gα12, Gα13 and Gαq. These studies point out that the above-mentioned Gα subunits might be involved in the WNT/ß-catenin and WNT/calcium-signalling activity by WNT16 in osteoblasts, and for Gα12 in its WNT/PCP-signalling activity, illustrating a novel possible mechanism of interplay between the different WNT-signalling pathways in osteoblasts. Additional studies are needed to demonstrate whether this mechanism is specific for WNT16 signalling or relevant for all other WNT ligands as well. Altogether, we further defined WNT16's mechanism of action in osteoblasts that might underlie the well-known beneficial effects of WNT16 on skeletal homeostasis. These findings on WNT16 and the activity of specific Gα subunits in osteoblasts could definitely contribute to the development of novel therapeutic approaches for fragility fractures in the future.

Camurati-Engelmann Disease.

Camurati-Engelmann disease or progressive diaphyseal dysplasia is a rare autosomal dominant sclerosing bone dysplasia. Mainly the skull and the diaphyses of the long tubular bones are affected. Clinically, the patients suffer from bone pain, easy fatigability, and decreased muscle mass and weakness in the proximal parts of the lower limbs resulting in gait disturbances. The disease-causing mutations are located within the TGFß-1 gene and expected to or thought to disrupt the binding between TGFß1 and its latency-associated peptide resulting in an increased signaling of the pathway and subsequently accelerated bone turnover. In preclinical studies, it was shown that targeting the type I receptor ameliorates the high bone turnover. In patients, treatment options are currently mostly limited to corticosteroids that may relieve the pain, and improve the muscle weakness and fatigue. In this review, the clinical and radiological characteristics as well as the molecular genetics of this condition are discussed.

Genetic Variation in RIN3 in the Belgian Population Supports Its Involvement in the Pathogenesis of Paget's Disease of Bone and Modifies the Age of Onset.

Paget's disease of bone (PDB) is a common, late-onset bone disorder characterized by focal increase of bone turnover. Mutations in the SQSTM1 gene are found in up to 40% of patients and recent GWAS have led to novel associations with several loci. RIN3, the candidate gene located at the associated 14q32 locus, has recently been studied in a British cohort to elucidate its contribution to the pathogenesis. In this study, we performed a genetic screening of RIN3 in an unrelated cohort to validate these findings and to further explore genetic variation in this gene in the context of PDB. In our screening, we examined the 5' untranslated region (UTR), the exonic regions and the intron-exon boundaries of the gene in a control cohort and a patient cohort. Our findings show clustering of variation similar to the British cohort and support a protective role for common genetic variation (rs117068593, p.R279C) in the proline-rich region and a functionally relevant role for rare genetic variation in the domains that mediate binding and activation of its interaction partner, Rab5. Additive regression models, fitted for the common variants, validated the association of the rs117068593 variant with the disease (OR+/+ 0.315; OR+/- 0.562). In addition, our analyses revealed a potentially modifying effect of this variant on the age of onset of the disease. In conclusion, our findings support the involvement of genetic variation in RIN3 in PDB and suggest a role for RIN3 as a potential modifier of the age of onset of the disease.

Human Genetics of Sclerosing Bone Disorders.

PURPOSE OF REVIEW: The group of sclerosing bone disorders encompasses a variety of disorders all marked by increased bone mass. In this review, we give an overview of the genetic causes of this heterogeneous group of disorders and briefly touch upon the value of these findings for the development of novel therapeutic agents. RECENT FINDINGS: Advances in the next-generation sequencing technologies are accelerating the molecular dissection of the pathogenic mechanisms underlying skeletal dysplasias. Throughout the years, the genetic cause of these disorders has been extensively studied which resulted in the identification of a variety of disease-causing genes and pathways that are involved in bone formation by osteoblasts, bone resorption by osteoclasts, or both processes. Due to this rapidly increasing knowledge, the insights into the regulatory mechanisms of bone metabolism are continuously improving resulting in the identification of novel therapeutic targets for disorders with reduced bone mass and increased bone fragility.

Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta.

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

Genetic Screening of WNT4 and WNT5B in Two Populations with Deviating Bone Mineral Densities.

A role for WNT4 and WNT5B in bone metabolism was indicated by genome-wide association studies (GWAS) and a Wnt4 knockout mouse model. The aim of this study was therefore to replicate and further investigate the causality between genetic variation in WNT4 and WNT5B and deviating bone mineral density (BMD) values. A WNT4 and WNT5B mutation screening was performed in patients with craniotubular hyperostosis using Sanger sequencing. Here, no putative causal mutations were detected. Moreover, a high and low BMD cohort was selected from the Odense Androgen Study population for re-sequencing. In WNT4 we detected four variants (three rare, one common), while in WNT5B we detected five variants (two rare, three common). For the common variants, no significant difference in genotype frequencies between the high and low BMD cohorts was observed. The SNPs associated with the GWAS were genotyped in these cohorts, but again no significant difference in genotype frequencies was observed. Despite the findings of the GWAS, we were not able to replicate or further verify the genetic association of polymorphisms in WNT4 and WNT5B with BMD. In order to do so, the intronic regions of both genes could be investigated more thoroughly in more extended populations (or extremes) with greater power. Future genetic and functional studies toward adjacent genes of WNT4 and WNT5B can also be interesting to figure out whether the signal from GWAS could possibly be attributed to genetic variation in these genes.

Sclerosing bone dysplasias: leads toward novel osteoporosis treatments.

Sclerosing bone dysplasias are a group of rare, monogenic disorders characterized by increased bone density resulting from the disturbance in the fragile equilibrium between bone formation and resorption. Over the last decade, major contributions have been made toward better understanding of the pathogenesis of these conditions. These studies provided us with important insights into the bone biology and yielded the identification of numerous drug targets for the prevention and treatment of osteoporosis. Here, we review this heterogeneous group of disorders focusing on their utility in the development of novel osteoporosis therapies.

Resistin polymorphisms show associations with obesity, but not with bone parameters in men: results from the Odense Androgen Study.

Resistin is an obesity-related adipokine which has also been implicated in bone metabolism. Therefore, we designed a study to investigate the possible role of resistin gene variation in both obesity and bone mineral density. We included 1,155 individuals from the Odense Androgen Study (663 young subjects and 492 older subjects), a population-based, prospective, observational study on the inter-relationship between endocrine status, body composition, muscle function, and bone metabolism in men, in an association study with resistin (RETN) polymorphisms. Three RETN variants (rs1862513, rs3745367 and rs3745369) were genotyped with TaqMan Pre-Designed Genotyping assays. Linear regression was performed to investigate the possible association of these variants with several obesity- and bone-related parameters. After genotyping 1,155 Danish men, 663 young subjects and 492 older subjects, we found that rs3745367 was associated with several obesity-related measures in both the young and elderly cohort. Rs3745369 was only associated with obesity-phenotypes in the elderly cohort. When studying the combined cohorts, we could confirm the associations of rs3745367 with several obesity-related parameters. We were unable to identify any association between RETN polymorphisms and bone-related measurements. Together, these results illustrate resistin's role in the development of obesity. Rs3745367 gives the most consistent results in the current study and these should be confirmed in other populations. Research into its possible functional effect might also be required. A role for RETN variants in determining bone mineral density seems unlikely from our results.

Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.

Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/ß-catenin signaling. We found the extracellular ß-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.

Association study of common variants in the sFRP1 gene region and parameters of bone strength and body composition in two independent healthy Caucasian male cohorts.

Bone mineral density (BMD) and bone strength are predictive parameters for the development of osteoporosis and related fracture later in life. Although it is well known that BMD and bone strength have a high heritability, not much of the variation is already explained. Mice models showed that sFRP1 has an influence on bone formation. Therefore this study aimed to investigate the effect of common genetic variation on BMD and bone strength in Caucasian men of different ages. Using HapMap we selected 13 tagSNPs which tag most common genetic variation in and around sFRP1 and we genotyped these SNPs in the young cohort of the Odense Androgen Study (OAS). The OAS includes a total of 1383 Danish men from two different age groups ([20-29 years]: N=783; [60-74 years]: N=600) and is well characterised. The subjects were phenotyped for BMD at several sites, and additionally for body composition and hip geometry parameters. Based on the results of the young cohort we selected three SNPs for further analysis in the complete OAS population. To conclude we tried to replicate the results of two SNPs in an independent population of 994 Belgian men. We found a strong association for rs9694405 with BMI as well in both cohorts separately as in the whole OAS population. Further we found rs4736965 associated with several hip geometry parameters in the same population. However we were not able to replicate those results in the Belgian population. At last we found in the OAS population age specific effects for rs10106678 with whole body BMD and waist to hip ratio.

Single nucleotide polymorphisms in sFRP4 are associated with bone and body composition related parameters in Danish but not in Belgian men.

The senescence accelerated mouse P6 (SAMP6) has a low bone mass and has previously shown to be a good model for senile osteoporosis in humans. In addition to a reduced bone mass, SAMP6 mice are obese and have hyperlipidemia. Using positional cloning and expression studies, an increased expression of sfrp4 was found in these mice. SFRP4 is a modulator of the Wnt signalling pathway. This pathway has been previously shown to be involved in regulating bone mass. Additional evidence that sFRP4 has an influence on BMD was delivered by linkage and association studies mostly performed in Asian populations. Based on these data we decided to perform an association study between common variants in sFRP4, BMD, hip geometry parameters and body composition parameters in a population consisting of 1383 Danish men (783 aged 20-29 years; 600 aged 60-74 years). Afterwards we tried to replicate the significant results in a population of 994 Belgian men. In the Danish population we found 6 SNPs associated with BMD at the hip and/or femoral neck. Furthermore, all 6 SNPs were associated with several hip geometry parameters. The homozygous presence of the minor allele resulted for all SNPs (except rs4720265) in a decrease in bone density and bone strength. Finally, we observed in the Danish population age specific associations with height and fat mass. In the Belgian population we tried to replicate the results of three SNPs with BMD and body composition parameters. Unfortunately, we were not able to replicate the results found in the Danish cohort but we found one SNP (rs2598116) associated with height. In conclusion, genetic variation in sFRP4 has an influence on hip fracture risk, percentage body fat and height in a Danish male population. However, we were unable to replicate these results in an independent Belgian population.

Broadening the Spectrum of Loss-of-Function Variants in NPR-C-Related Extreme Tall Stature.

CONTEXT: Natriuretic peptide receptor-C (NPR-C, encoded by NPR3) belongs to a family of cell membrane-integral proteins implicated in various physiological processes, including longitudinal bone growth. NPR-C acts as a clearance receptor of natriuretic peptides, including C-type natriuretic peptide (CNP), that stimulate the cGMP-forming guanylyl cyclase-coupled receptors NPR-A and NPR-B. Pathogenic variants in CNP, NPR2, and NPR3 may cause a tall stature phenotype associated with macrodactyly of the halluces and epiphyseal dysplasia. OBJECTIVE: Here we report on a boy with 2 novel biallelic inactivating variants of NPR3. METHODS: History and clinical characteristics were collected. Biochemical indices of natriuretic peptide clearance and in vitro cellular localization of NPR-C were studied to investigate causality of the identified variants. RESULTS: We identified 2 novel compound heterozygous NPR3 variants c.943G>A p.(Ala315Thr) and c.1294A>T p.(Ile432Phe) in a boy with tall stature and macrodactyly of the halluces. In silico analysis indicated decreased stability of NPR-C, presumably resulting in increased degradation or trafficking defects. Compared to other patients with NPR-C loss-of-function, the phenotype seemed to be milder: pseudo-epiphyses in hands and feet were absent, biochemical features were less severe, and there was some co-localization of p.(Ile432Phe) NPR-C with the cell membrane, as opposed to complete cytoplasmic retention. CONCLUSION: With this report on a boy with tall stature and macrodactyly of the halluces we further broaden the genotypic and phenotypic spectrum of NPR-C-related tall stature.

Identification of Compound Heterozygous Variants in LRP4 Demonstrates That a Pathogenic Variant outside the Third ß-Propeller Domain Can Cause Sclerosteosis.

Sclerosteosis is a high bone mass disorder, caused by pathogenic variants in the genes encoding sclerostin or LRP4. Both proteins form a complex that strongly inhibits canonical WNT signaling activity, a pathway of major importance in bone formation. So far, all reported disease-causing variants are located in the third ß-propeller domain of LRP4, which is essential for the interaction with sclerostin. Here, we report the identification of two compound heterozygous variants, a known p.Arg1170Gln and a novel p.Arg632His variant, in a patient with a sclerosteosis phenotype. Interestingly, the novel variant is located in the first ß-propeller domain, which is known to be indispensable for the interaction with agrin. However, using luciferase reporter assays, we demonstrated that both the p.Arg1170Gln and the p.Arg632His variant in LRP4 reduced the inhibitory capacity of sclerostin on canonical WNT signaling activity. In conclusion, this study is the first to demonstrate that a pathogenic variant in the first ß-propeller domain of LRP4 can contribute to the development of sclerosteosis, which broadens the mutational spectrum of the disorder.