Bone development and remodeling in metabolic disorders

There are many metabolic disorders that present with bone phenotypes. In some cases, the pathological bone symptoms are the main features of the disease whereas in others they are a secondary characteristic. In general, the generation of the bone problems in these disorders is not well understood and the therapeutic options for them are scarce. Bone development occurs in the early stages of embryonic development where the bone formation, or osteogenesis, takes place. This osteogenesis can be produced through the direct transformation of the pre-existing mesenchymal cells into bone tissue (intramembranous ossification) or by the replacement of the cartilage by bone (endochondral ossification). In contrast, bone remodeling takes place during the bone's growth, after the bone development, and continues throughout the whole life. The remodeling involves the removal of mineralized bone by osteoclasts followed by the formation of bone matrix by the osteoblasts, which subsequently becomes mineralized. In some metabolic diseases, bone pathological features are associated with bone development problems but in others they are associated with bone remodeling. Here, we describe three examples of impaired bone development or remodeling in metabolic diseases, including work by others and the results from our research. In particular, we will focus on hereditary multiple exostosis (or osteochondromatosis), Gaucher disease, and the susceptibility to atypical femoral fracture in patients treated with bisphosphonates for several years

Genetics and Genomics of SOST: functional analysis of variants and genomic regulation in osteoblasts

SOST encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the SOST proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the SOST proximal promoter and the ECR5 enhancer, several additional enhancers located between EVT4 and MEOX1 and a distant region containing exon 18 of DHX8. In conclusion, SOST presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells

Gene Network of Susceptibility to Atypical Femoral Fractures Related to Bisphosphonate Treatment

Atypical femoral fractures (AFF) are rare fragility fractures in the subtrocantheric or diaphysis femoral region associated with long-term bisphosphonate (BP) treatment. The etiology of AFF is still unclear even though a genetic basis is suggested. We performed whole exome sequencing (WES) analysis of 12 patients receiving BPs for at least 5 years who sustained AFFs and 4 controls, also long-term treated with BPs but without any fracture. After filtration and prioritization of rare variants predicted to be damaging and present in genes shared among at least two patients, a total of 272 variants in 132 genes were identified. Twelve of these genes were known to be involved in bone metabolism and/or AFF, highlighting DAAM2 and LRP5, both involved in the Wnt pathway, as the most representative. Afterwards, we intersected all mutated genes with a list of 34 genes obtained from a previous study of three sisters with BP-related AFF, identifying nine genes. One of these (MEX3D) harbored damaging variants in two AFF patients from the present study and one shared among the three sisters. Gene interaction analysis using the AFFNET web suggested a complex network among bone-related genes as well as with other mutated genes. BinGO biological function analysis highlighted cytoskeleton and cilium organization. In conclusion, several genes and their interactions could provide genetic susceptibility to AFF, that along with BPs treatment and in some cases with glucocorticoids may trigger this so feared complication

Functional characterization of a GGPPS variant indentified in atypical femoral fracture patients and delineation of the role of GGPPS in bone-relevant cell types

Atypical femoral fractures (AFFs) are a rare but potentially devastating event, often but not always linked to bisphosphonate (BP) therapy. The pathogenic mechanisms underlying AFFs remain obscure, and there are no tests available that might assist in identifying those at high risk of AFF. We previously used exome sequencing to explore the genetic background of three sisters with AFFs and three additional unrelated AFF cases, all previously treated with BPs. We detected 37 rare mutations (in 34 genes) shared by the three sisters. Notably, we found a p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase, a component of the mevalonate pathway, which is critical to osteoclast function and is inhibited by N-BPs. In addition, the CYP1A1 gene, responsible for the hydroxylation of 17β-estradiol, estrone, and vitamin D, was also mutated in all three sisters and one unrelated patient. Here we present a detailed list of the variants found and report functional analyses of the GGPS1 p.Asp188Tyr mutation, which showed a severe reduction in enzyme activity together with oligomerization defects. Unlike BP treatment, this genetic mutation will affect all cells in the carriers. RNAi knockdown of GGPS1 in osteoblasts produced a strong mineralization reduction and a reduced expression of osteocalcin, osterix, and RANKL, whereas in osteoclasts, it led to a lower resorption activity. Taken together, the impact of the mutated GGPPS and the relevance of the downstream effects in bone cells make it a strong candidate for AFF susceptibility. We speculate that other genes such as CYP1A1 might be involved in AFF pathogenesis, which remains to be functionally proved. The identification of the genetic background for AFFs provides new insights for future development of novel risk assessment tools. © 2018 American Society for Bone and Mineral Research

GGPS1 Mutation and Atypical Femoral Fractures with Bisphosphonates

Atypical femoral fractures have been associated with long-term bisphosphonate treatment.1,2 However, the underlying mechanisms remain obscure. We studied three sisters who had atypical femoral fractures after receiving various oral bisphosphonates for 6 years. Two of the sisters had a single fracture (at the ages of 64 and 73 years), and one had bilateral fractures (one at the age of 60 years and the other at the age of 61 years). Given the low incidence of atypical femoral fractures in the general population (5.9 per 10,000 person-years),3 we hypothesized that these sisters might have an underlying genetic background that contributed to these fractures

Identification and functional characterization of genetic loci involved in osteoporosis and atypical femoral fracture

[eng] Osteoporosis is a complex disease, determined by genetic and environmental factors, characterized by a low bone mass and microarchitectural deterioration of bone tissue, with a consequent decrease of bone strength and increase in bone fragility and fracture risk. It is the most common bone metabolic disorder, and a major worldwide public health concern. Bone mineral density (BMD) heritability ranges from 50% to 85% and genetic factors have a crucial role in the pathogenesis of osteoporosis. To date, many genes and variants associated with osteoporosis have been identified, but they only explain 20% of the phenotypic variance. In addition, the underlying mechanisms of these associations are poorly understood. Hence, it is necessary to identify new variants/genes and to deepen the knowledge about osteoporosis pathogenesis. Nitrogen-containing bisphosphonates (N-BPs) are the first-line pharmacological treatment for osteoporosis. They are cost-effective anti-resorptive drugs that inhibit the mevalonate pathway, preventing osteoclasts’ function and promoting their apoptosis. Very rarely, atypical femoral fractures (AFFs) can occur after a long-term therapy with N-BPs. AFFs are located in the subtrochanteric region or the femoral diaphysis and arise after minimal or no trauma. The pathogenic mechanisms underlying AFF remain largely unknown. This PhD thesis aimed at contributing to the elucidation of the genetic determinants of osteoporosis and AFF. On the one side, we deeply studied a GWAS signal in the C7ORF76 locus, in 7q21.3, including its dissection in the BARCOS cohort of Spanish postmenopausal women and the functional characterization of the associated variants and regulatory elements within the locus. We identified 2 variants associated with BMD and osteoporotic fracture and showed that they are cis-eQTLs for the neighbouring gene SLC25A13 in human primary osteoblasts. One of the associated variants lay in an upstream putative regulatory element, which we named UPE. We have demonstrated the regulatory capacity of UPE in bone cells, and its interaction with a lncRNA and other regulatory elements within the region. We also studied a previously described mouse Dlx5/6 enhancer (eDlx#18) within the C7ORF76 locus. We demonstrated that it is able to activate transcription in an osteoblastic context and that it interacts with the DLX5 promoter, as well as with other tissue-specific DLX5/6 enhancers. An SNP within this enhancer has been shown to be a cis-eQTL for DLX6 in human primary osteoblasts. Finally, the homozygous deletion of eDlx#18 in mice resulted in a reduced viability, a decreased Dlx5 expression in otic vesicle and branchial arches in E11.5 embryos. In E17.5 embryos, skeletal defects were noted, including a smaller dentary and deficient ossification at several sites. On the other side, a small cohort of N-BP-associated AFF patients was analyzed by whole exome sequencing, and the most conserved and interesting mutation found, in the GGPS1 gene, was functionally characterized using molecular and cellular approaches. We found 37 rare mutations in 34 genes shared by 3 sisters with N-BP-associated AFF, including mutations in GGPS1 and CYP1A1, which was also mutated in one unrelated patient. The p.Asp188Tyr mutation in GGPS1 affected oligomerization of the enzyme, leading to a severe reduction in enzyme activity. GGPS1 depletion in osteoblastic cells resulted in a strong mineralization reduction and a decreased expression of some osteoblastic markers, such as osteocalcin, osterix and RANKL. The depletion in osteoclast precursors led to increased osteoclast number but with reduced resorption activity

Gene network of susceptibility to atypical femoral fractures related to bisphosphonate treatment

Atypical femoral fractures (AFF) are rare fragility fractures in the subtrocantheric or diaphysis femoral region associated with long-term bisphosphonate (BP) treatment. The etiology of AFF is still unclear even though a genetic basis is suggested. We performed whole exome sequencing (WES) analysis of 12 patients receiving BPs for at least 5 years who sustained AFFs and 4 controls, also long-term treated with BPs but without any fracture. After filtration and prioritization of rare variants predicted to be damaging and present in genes shared among at least two patients, a total of 272 variants in 132 genes were identified. Twelve of these genes were known to be involved in bone metabolism and/or AFF, highlighting DAAM2 and LRP5, both involved in the Wnt pathway, as the most representative. Afterwards, we intersected all mutated genes with a list of 34 genes obtained from a previous study of three sisters with BP-related AFF, identifying nine genes. One of these (MEX3D) harbored damaging variants in two AFF patients from the present study and one shared among the three sisters. Gene interaction analysis using the AFFNET web suggested a complex network among bone-related genes as well as with other mutated genes. BinGO biological function analysis highlighted cytoskeleton and cilium organization. In conclusion, several genes and their interactions could provide genetic susceptibility to AFF, that along with BPs treatment and in some cases with glucocorticoids may trigger this so feared complication.This research was funded by MCIN/AEI/10.13039/501100011033, Project PID2019-107188RB-C21. The research was also supported by Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (grants number CB16/10/00245); Fundación Española de Investigación Ósea y del Metabolismo Mineral (FEIOMM); Formación en Investigación en Salud (grant number PI19/00033) from Instituto de Salud Carlos III (ISCIII); and European Regional Development Fund. DO is recipient of a Sara Borrell grant from ISCIII

Common and rare variants of WNT16, DKK1 and SOST and their relationship with bone mineral density

Numerous GWAS and candidate gene studies have highlighted the role of the Wnt pathway in bone biology. Our objective has been to study in detail the allelic architecture of three Wnt pathway genes: WNT16, DKK1 and SOST, in the context of osteoporosis. We have resequenced the coding and some regulatory regions of these three genes in two groups with extreme bone mineral density (BMD) (n=∼50, each) from the BARCOS cohort. No interesting novel variants were identifed. Thirteen predicted functional variants have been genotyped in the full cohort (n=1490), and for ten of them (with MAF>0.01), the association with BMD has been studied. We have found six variants nominally associated with BMD, of which 2 WNT16 variants predicted to be eQTLs for FAM3C (rs55710688, in the Kozak sequence and rs142005327, within a putative enhancer) withstood multiple-testing correction. In addition, two rare variants in functional regions (rs190011371 in WNT16b 3′UTR and rs570754792 in the SOST TATA box) were found only present in three women each, all with BMD below the mean of the cohort. Our results reinforce the higher importance of regulatory versus coding variants in these Wnt pathway genes and open new ways for functional studies of the relevant variants

Functional characterization of the C7ORF76 genomic region, a prominent GWAS signal for osteoporosis in 7q21.3

Genome-wide association studies (GWAS) have repeatedly identified genetic variants associated with bone mineral density (BMD) and osteoporotic fracture in non-coding regions of C7ORF76, a poorly studied gene of unknown function. The aim of the present study was to elucidate the causality and molecular mechanisms underlying the association. We re-sequenced the genomic region in two extreme BMD groups from the BARCOS cohort of postmenopausal women to search for functionally relevant variants. Eight selected variants were tested for association in the complete cohort and 2 of them (rs4342521 and rs10085588) were found significantly associated with lumbar spine BMD and nominally associated with osteoporotic fracture. cis-eQTL analyses of these 2 SNPs, together with SNP rs4727338 (GWAS lead SNP in Estrada et al., Nat Genet. 44:491-501, 2012), performed in human primary osteoblasts, disclosed a statistically significant influence on the expression of the proximal neighbouring gene SLC25A13 and a tendency on the distal SHFM1. We then studied the functionality of a putative upstream regulatory element (UPE), containing rs10085588. Luciferase reporter assays showed transactivation capability with a strong allele-dependent effect. Finally, 4C-seq experiments in osteoblastic cell lines showed that the UPE interacted with different tissue-specific enhancers and a lncRNA (LOC100506136) in the region. In summary, this study is the first one to analyse in depth the functionality of C7ORF76 genomic region. We provide functional regulatory evidence for the rs10085588, which may be a causal SNP within the 7q21.3 GWAS signal for osteoporosis.This work was supported by the following grants: SAF2014-56562-R, SAF2016-75948-R (Spanish MINECO), 2014SGR932 (Generalitat de Catalunya), CIBERER (U720), and FEIOMM Investigación 2014. NRA is a recipient of an FPU predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte; NMG is a recipient of a FI predoctoral fellowship from Generalitat de Catalunya

Functional analyses of four CYP1A1 missense mutations present in patients with atypical femoral fractures

Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.Funding was available from Fundación Española de Investigación Ósea y Metabolismo Mineral (“Beca FEIOMM 2019 de Investigación Básica”) and grant PID2019-107188RB-C21 from the Spanish Ministerio de Ciencia e Innovación. N.M.G. and N.R.A. were recipients of Ph.D. fellowships from AGAUR (Catalan Government) and the Spanish Ministerio de Universidades