Evolutionary and functional analyses of LRP5 in archaic and extant modern humans.

BACKGROUND: The human lineage has undergone a postcranial skeleton gracilization (i.e. lower bone mass and strength relative to body size) compared to other primates and archaic populations such as the Neanderthals. This gracilization has been traditionally explained by differences in the mechanical load that our ancestors exercised. However, there is growing evidence that gracilization could also be genetically influenced. RESULTS: We have analyzed the LRP5 gene, which is known to be associated with high bone mineral density conditions, from an evolutionary and functional point of view. Taking advantage of the published genomes of archaic Homo populations, our results suggest that this gene has a complex evolutionary history both between archaic and living humans and within living human populations. In particular, we identified the presence of different selective pressures in archaics and extant modern humans, as well as evidence of positive selection in the African and South East Asian populations from the 1000 Genomes Project. Furthermore, we observed a very limited evidence of archaic introgression in this gene (only at three haplotypes of East Asian ancestry out of the 1000 Genomes), compatible with a general erasing of the fingerprint of archaic introgression due to functional differences in archaics compared to extant modern humans. In agreement with this hypothesis, we observed private mutations in the archaic genomes that we experimentally validated as putatively increasing bone mineral density. In particular, four of five archaic missense mutations affecting the first ß-propeller of LRP5 displayed enhanced Wnt pathway activation, of which two also displayed reduced negative regulation. CONCLUSIONS: In summary, these data suggest a genetic component contributing to the understanding of skeletal differences between extant modern humans and archaic Homo populations.

Genetic Analysis in a Familial Case With High Bone Mineral Density Suggests Additive Effects at Two Loci.

Osteoporosis is the most common bone disease, characterized by a low bone mineral density (BMD) and increased risk of fracture. At the other end of the BMD spectrum, some individuals present strong, fracture-resistant, bones. Both osteoporosis and high BMD are heritable and their genetic architecture encompasses polygenic inheritance of common variants and some cases of monogenic highly penetrant variants in causal genes. We have investigated the genetics of high BMD in a family segregating this trait in an apparently Mendelian dominant pattern. We searched for rare causal variants by whole-exome sequencing in three affected and three nonaffected family members. Using this approach, we have identified 38 rare coding variants present in the proband and absent in the three individuals with normal BMD. Although we have found four variants shared by the three affected members of the family, we have not been able to relate any of these to the high-BMD phenotype. In contrast, we have identified missense variants in two genes, VAV3 and ADGRE5, each shared by two of out of three affected members, whose loss of function fits with the phenotype of the family. In particular, the proband, a woman displaying the highest BMD (sum Z-score = 7), carries both variants, whereas the other two affected members carry one each. VAV3 encodes a guanine-nucleotide-exchange factor with an important role in osteoclast activation and function. Although no previous cases of VAV3 mutations have been reported in humans, Vav3 knockout (KO) mice display dense bones, similarly to the high-BMD phenotype present in our family. The ADGRE5 gene encodes an adhesion G protein-coupled receptor expressed in osteoclasts whose KO mouse displays increased trabecular bone volume. Combined, these mouse and human data highlight VAV3 and ADGRE5 as novel putative high-BMD genes with additive effects, and potential therapeutic targets for osteoporosis. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

On the association between Chiari malformation type 1, bone mineral density and bone related genes.

Background: Chiari malformation type 1 (C1M) is a neurological disease characterized by herniation of the cerebellar tonsils below the foramen magnum. Cranial bone constriction is suspected to be its main cause. To date, genes related to bone development (e.g. DKK1 or COL1A2) have been associated with C1M, while some bone diseases (e.g. Paget) have been found to cosegregate with C1M. Nevertheless, the association between bone mineral density (BMD) and C1M has not been investigated, yet. Here, we systematically investigate the association between C1M and BMD, and between bone related genes and C1M. Methods: We have recruited a small cohort of C1M patients (12 unrelated patients) in whom we have performed targeted sequencing of an in-house bone-related gene panel and BMD determination through non-invasive DXA. Results: In the search for association between the bone related genes and C1M we have found variants in more than one C1M patient in WNT16, CRTAP, MYO7A and NOTCH2. These genes have been either associated with craniofacial development in different ways, or previously associated with C1M (MYO7A). Regarding the potential link between BMD and C1M, we have found three osteoporotic patients and one patient who had high BMD, very close to the HBM phenotype values, although most patients had normal BMD. Conclusions: Variants in bone related genes have been repeatedly found in some C1M cases. The relationship of bone genes with C1M deserves further study, to get a clearer estimate of their contribution to its etiology. No direct correlation between BMD and C1M was observed.

Functional Assessment of Coding and Regulatory Variants From the DKK1 Locus.

The DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome-wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt-pathway-specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high-bone-mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture- (4C-) sequencing experiment, we have detected that the region containing 24 BMD-GWA variants, located 350-kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA-activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss-of-function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long-range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Bone tissue quality in patients with monoclonal gammopathy of uncertain significance.

INTRODUCTION: Monoclonal gammopathy of uncertain significance (MGUS) is highly prevalent in older adults and affects bone structure, with osteoporosis and increased risk of fractures in up to 14% of affected patients. Dual-energy X-ray absorptiometry (DXA), the standard technique for diagnosing osteoporosis, is ineffective to reveal microstructure and bone quality in this disease. MATERIALS AND METHODS: We conducted a cross-sectional study of patients with MGUS, recruited consecutively from the Hematology and Internal Medicine Departments of Hospital del Mar, Barcelona, between January 2011 and January 2018. Medical records, clinical results and spinal X-ray images were collected. Bone mineral density (BMD) at hip and spine was measured by DXA and Bone Material Strength index (BMSi) by impact microindentation on the tibial mid-shaft. RESULTS: Thirty-nine patients with MGUS and 65 age-matched controls without previous fractures were included. In the MGUS group, 11 (28.2%) patients had prevalent fractures, nearly half of them vertebral (n = 5, 45.45%). Compared to controls, MGUS patients had significantly lower BMSi, a mean (SD) of 70.72 (9.70) vs. 78.29 (8.70), p = 0.001, and lower spinal BMD values (0.900 [0.159] vs. 1.003 [0.168], respectively, p = 0.012), but no significant differences at femoral neck and total hip. No association was observed between BMSi and DXA. Bone remodeling markers (procollagen type-1 N propeptide, bone-alkaline phosphatase and C-terminal telopeptide of type I collagen) did not differ between the two groups. CONCLUSIONS: Spinal BMD and mechanical properties of bone tissue, as measured by impact microindentation, were impaired in patients with MGUS. These changes in bone tissue mechanical resistance were independent of DXA levels.

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.

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 identified. 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.

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

Cortical and Trabecular Bone Analysis of Patients With High Bone Mass From the Barcelona Osteoporosis Cohort Using 3-Dimensional Dual-Energy X-ray Absorptiometry: A Case-Control Study.

High bone mass (HBM), a rare phenotype, can be detected by dual-energy X-ray absorptiometry (DXA) scanning. Measurements with peripheral quantitative computed tomography at the tibia have found increased trabecular bone mineral density and changes in cortical bone density and structure, all of which lead to increased bone strength. However, no studies on cortical and trabecular bone have been performed at the femur. The recently developed 3-dimensional (3D)-DXA software algorithm quantifies the trabecular and cortical volumetric bone mineral density (vBMD) and the anatomical distribution of cortical thickness using routine hip DXA scans. We analyzed the femurs of 15 women with HBM and 15 controls from the Barcelona Osteoporosis (BARCOS) cohort using the 3D-DXA technique. The mean vBMD of proximal femur was 29.7% higher in HBM cases than in controls for the integral bone, 41.3% higher for the trabecular bone, and 7.3% higher for the cortical bone (p < 0.001). No differences in bone size were detected between cases and controls. Patients with HBM had a thicker cortex and higher trabecular and cortical vBMDs, as measured by 3D-DXA at the femur and compared to controls; bone size was similar in both groups. To the best of our knowledge, this is the first description of trabecular and cortical characteristics of the hip in patients with HBM.

Fracture during oral bisphosphonate therapy is associated with deteriorated bone material strength index.

BACKGROUND: Some patients experience fractures while receiving oral bisphosphonates (BPs) treatment. Clinical risk factors, advanced bone density loss, and microarchitecture deterioration have been associated with such fractures but bone tissue properties other than bone mineral density (BMD) have not been assessed. METHODS: In a cross-sectional study of postmenopausal women on bisphosphonates for at least 4years with good adherence to treatment, 21 patients with incident fractures were compared with 18 treated patients without new fractures. Demographic and clinical variables, BMD, laboratory tests, and bone material strength index (BMSi) assessed by impact microindentation at the tibial diaphysis were recorded for all participants. RESULTS: Clinical and laboratory results did not differ between patients taking BPs with incident fractures and those without new fractures. However, BMSi was significantly lower (mean±SD) in those who fractured (73.76±6.49) than in no-fracture patients (81.64±6.26; p=0.001). Lumbar spine (LS) BMD was also lower in fractured patients (p=0.03). Adjusted models including age, body mass index, years on BP treatment, and LS-BMD confirmed an increase in fracture risk per BMSi standard deviation decrease: adjusted OR 23.5 [95% CI 2.16 to 255.66], p=0.01. ROC analyses showed an area under the curve of 0.82 (95% CI 0.68 to 0.95) for BMSi, higher than that for BMD at any location, which ranged from 0.64 (95% CI 0.47 to 0.82) for femoral neck (FN) BMD to 0.71 (95% CI 0.55 to 0.87) for LS-BMD. CONCLUSIONS: Patients who fracture while receiving BPs treatment have worse BMSi scores than BP-treated patients without fractures. The potential for BMSi to provide an additional osteoporosis treatment target should be explored.

Mujer de 31 años con lesiones osteolíticas múltiples asociadas a afectación linfática tisular / 31 years old woman with multiple osteolytic lesions associated with lymphatic tissue damage

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Brief Report: HIV Infection Is Associated With Worse Bone Material Properties, Independently of Bone Mineral Density.

Low bone mineral density (BMD) in HIV-infected individuals has been documented in an increasing number of studies. However, it is not clear whether it is the infection itself or the treatment that causes bone impairment. Microindentation measures bone material strength (Bone Material Strength index) directly. We recruited 85 patients, 50 infected with HIV and 35 controls. Median Bone Material Strength index was 84.5 (interquartile range 83-87) in HIV-infected patients and 90 (88.5-93) in controls (P < 0.001). No significant differences in BMD between cases and controls at any of the sites examined (total hip, femoral neck, and lumbar spine). HIV infection is associated with bone damage, independently of BMD.

MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones.

BACKGROUND: MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. METHODS: Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). RESULTS: Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. CONCLUSIONS: We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy.

Bone Tissue Properties Measurement by Reference Point Indentation in Glucocorticoid-Induced Osteoporosis.

Glucocorticoids, widely used in inflammatory disorders, rapidly increase bone fragility and, therefore, fracture risk. However, common bone densitometry measurements are not sensitive enough to detect these changes. Moreover, densitometry only partially recognizes treatment-induced fracture reductions in osteoporosis. Here, we tested whether the reference point indentation technique could detect bone tissue property changes early after glucocorticoid treatment initiation. After initial laboratory and bone density measurements, patients were allocated into groups receiving calcium + vitamin D (Ca+D) supplements or anti-osteoporotic drugs (risedronate, denosumab, teriparatide). Reference point indentation was performed on the cortical bone layer of the tibia by a handheld device measuring bone material strength index (BMSi). Bone mineral density was measured by dual-energy X-ray absorptiometry (DXA). Although Ca+D-treated patients exhibited substantial and significant deterioration, risedronate-treated patients exhibited no significant change, and both denosumab- and teriparatide-treated participants exhibited significantly improved BMSi 7 weeks after initial treatment compared with baseline; these trends remained stable for 20 weeks. In contrast, no densitometry changes were observed during this study period. In conclusion, our study is the first to our knowledge to demonstrate that reference point indentation is sensitive enough to reflect changes in cortical bone indentation after treatment with osteoporosis therapies in patients newly exposed to glucocorticoids.

Genetic analysis of high bone mass cases from the BARCOS cohort of Spanish postmenopausal women.

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM.