Enzymatic and Non-enzymatic Collagen Cross-Links and Fracture Occurrence in Type 1 Diabetes Patients.

Increased fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD) by DXA. Advanced glycation end-products (AGEs) have been implicated in the increased fracture risk in T1D, yet recent publications question this. To test the hypothesis that enzymatic collagen cross-links rather than AGEs correlate with fracture incidence in T1D, we analyzed iliac crest biopsies from sex-matched, fracturing T1D patients (N = 5; T1DFx), 6 non-fracturing T1D patients (T1DNoFx), and 6 healthy subjects, by Raman microspectroscopy as a function of tissue age (based on double fluorescent labels), in intracortical and trabecular bone, to determine pyridinoline (Pyd), ε-N-Carboxymethyl-L-lysine, and pentosidine (PEN)). There were no differences in the clinical characteristics between the T1DFx and T1DNoFx groups. At trabecular forming surfaces, T1DFx patients had higher PEN and Pyd content compared to T1DNoFx ones. Previous studies have shown that elevated PEN does not necessarily correlate with fracture incidence in postmenopausal, long-term T1D patients. On the other hand, the elevated Pyd content in the T1DFx patients would be consistent with published studies showing a significant correlation between elevated trivalent enzymatic collagen cross-links and fracture occurrence independent of BMD. Collagen fibers with high Pyd content are more brittle. Thus, a plausible suggestion is that it is the enzymatic collagen cross-links that either by themselves or in combination with the adverse effects of increased AGE accumulation that result in fragility fracture in T1D.

No evidence of mineralization abnormalities in iliac bone of premenopausal women with type 2 diabetes mellitus.

OBJECTIVES: Patients with type-2 diabetes mellitus (T2DM) have increased risk for bone fractures which points towards impaired bone quality. METHODS: We measured bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics based on quantitative backscattered electron images of transiliac biopsy samples from n=26 premenopausal women with T2DM. Outcomes were compared to those from reference cohorts as well as between T2DM subgroups defined by clinical characteristics. RESULTS: Comparison to references did not reveal any differences in BMDD (all p>0.05) but a lowered OLS-density in cancellous bone in T2DM (-14.9%, p<0.001). Neither BMDD nor OLS-characteristics differed in T2DM subgroups defined by HbA1c (<7% versus >7%). The average degree of bone mineralization (CaMean) was higher (0.44 wt%Ca in T2DM, 0.30 wt%Ca in reference) and consistently the calcium concentration between the tetracycline double labels (CaYoung) was higher (0.76 wt%Ca, all p<0.001) in cancellous versus cortical bone. CONCLUSIONS: Our findings suggest that bone matrix mineralization was neither affected by the presence nor by the glycemic control of T2DM in our study cohort. The intra-individual differences between cancellous and cortical bone mineralization gave evidence for differences in the time course of the early mineralization process in these compartments in general.

Exploiting the WNT Signaling Pathway for Clinical Purposes.

PURPOSE OF REVIEW: The goal of this paper is to evaluate critically the literature published over the past 3 years regarding the Wnt signaling pathway. The Wnt pathway was found to be involved in bone biology in 2001-2002 with the discovery of a (G171V) mutation in the lipoprotein receptor-related protein 5 (LRP5) that resulted in high bone mass and another mutation that completely inactivated Lrp5 function and resulted in osteoporosis pseudoglioma syndrome (OPPG). The molecular biology has been complex, and very interesting. It has provided many opportunities for exploitation to develop new clinical treatments, particularly for osteoporosis. More clinical possibilities include: treatments for fracture healing, corticosteroid osteoporosis, osteogenesis imperfecta, and others. In addition, we wish to provide historical information coming from distant publications (~350 years ago) regarding bone biology that have been confirmed by study of Wnt signaling. RECENT FINDINGS: A recent finding is the development of an antibody to sclerostin that is under study as a treatment for osteoporosis. Development of treatments for other forms of osteoporosis, such as corticosteroid osteoporosis, is also underway. The full range of the applications of the work is not yet been achieved.

Oral ibandronate in postmenopausal osteoporotic women alters micromechanical properties independently of changes in mineralization.

Postmenopausal osteoporotic (PMOP) women treated with ibandronate had higher bone mineral density, lower bone turnover, and decreased incidence of new vertebral fractures. The aim of this study was to investigate the effect of daily or intermittent oral ibandronate on the degree of mineralization (DMB) of bone and microhardness (Hv) at the bone tissue and bone structural unit (BSU) levels. A total of 110 iliac biopsies were taken from patients treated for 22 or 34 months with an oral placebo (n = 36), 2.5 mg daily oral ibandronate (n = 40), or 20 mg intermittent oral ibandronate (n = 34). These regimens provide annual cumulative exposures (ACEs) that are about half of the therapeutic doses currently licensed for PMOP women. DMB and Hv were measured at the global level (i.e., cortical or cancellous) and the focal level (i.e., BSU). At the global level, DMB and its distribution were not significantly different from placebo after 22 and 34 months of treatment. Hv was significantly higher in the cortical, cancellous, and total bone after 22 and 34 months of ibandronate versus placebo for both regimens. At the focal level, DMB and Hv, measured simultaneously in 3,760 BSUs, were significantly and positively correlated in all groups (r = 0.59-0.65, p < 0.0001). However, analysis of covariance highlighted the differences in the y intercepts of the linear regressions of the placebo- and ibandronate-treated groups. We infer that a low ACE of oral ibandronate altered the bone micromechanical properties irrespective of changes in secondary mineralization.

Genome-wide association study identifies ALDH7A1 as a novel susceptibility gene for osteoporosis.

Osteoporosis is a major public health problem. It is mainly characterized by low bone mineral density (BMD) and/or low-trauma osteoporotic fractures (OF), both of which have strong genetic determination. The specific genes influencing these phenotypic traits, however, are largely unknown. Using the Affymetrix 500K array set, we performed a case-control genome-wide association study (GWAS) in 700 elderly Chinese Han subjects (350 with hip OF and 350 healthy matched controls). A follow-up replication study was conducted to validate our major GWAS findings in an independent Chinese sample containing 390 cases with hip OF and 516 controls. We found that a SNP, rs13182402 within the ALDH7A1 gene on chromosome 5q31, was strongly associated with OF with evidence combined GWAS and replication studies (P = 2.08x10(-9), odds ratio = 2.25). In order to explore the target risk factors and potential mechanism underlying hip OF risk, we further examined this candidate SNP's relevance to hip BMD both in Chinese and Caucasian populations involving 9,962 additional subjects. This SNP was confirmed as consistently associated with hip BMD even across ethnic boundaries, in both Chinese and Caucasians (combined P = 6.39x10(-6)), further attesting to its potential effect on osteoporosis. ALDH7A1 degrades and detoxifies acetaldehyde, which inhibits osteoblast proliferation and results in decreased bone formation. Our findings may provide new insights into the pathogenesis of osteoporosis.

Teriparatide Followed by Denosumab in Premenopausal Idiopathic Osteoporosis: Bone Microstructure and Strength by HR-pQCT.

Premenopausal women with idiopathic osteoporosis (PreMenIOP) have marked deficits in skeletal microstructure. We have reported that sequential treatment with teriparatide and denosumab improves central skeletal bone mineral density (BMD) by dual-energy X-ray absorptiometry and central QCT in PreMenIOP. We conducted preplanned analyses of high-resolution peripheral quantitative computed tomography (HR-pQCT) scans from teriparatide and denosumab extension studies to measure effects on volumetric BMD (vBMD), microarchitecture, and estimated strength at the distal radius and tibia. Of 41 women enrolled in the parent teriparatide study (20 mcg daily), 34 enrolled in the HR-pQCT study. HR-pQCT participants initially received teriparatide (N = 24) or placebo (N = 10) for 6 months; all then received teriparatide for 24 months. After teriparatide, 26 enrolled in the phase 2B denosumab extension (60 mg q6M) for 24 months. Primary outcomes were percentage change in vBMD, microstructure, and stiffness after teriparatide and after denosumab. Changes after sequential teriparatide and denosumab were secondary outcomes. After teriparatide, significant improvements were seen in tibial trabecular number (3.3%, p = 0.01), cortical area and thickness (both 2.7%, p < 0.001), and radial trabecular microarchitecture (number: 6.8%, thickness: 2.2%, separation: -5.1%, all p < 0.02). Despite increases in cortical porosity and decreases in cortical density, whole-bone stiffness and failure load increased at both sites. After denosumab, increases in total (3.5%, p < 0.001 and 3.3%, p = 0.02) and cortical vBMD (1.7% and 3.2%; both p < 0.01), and failure load (1.1% and 3.6%; both p < 0.05) were seen at tibia and radius, respectively. Trabecular density (3.5%, p < 0.001) and number (2.4%, p = 0.03) increased at the tibia, while thickness (3.0%, p = 0.02) increased at the radius. After 48 months of sequential treatment, significant increases in total vBMD (tibia: p < 0.001; radius: p = 0.01), trabecular microstructure (p < 0.05), cortical thickness (tibia: p < 0.001; radius: p = 0.02), and whole bone strength (p < 0.02) were seen at both sites. Significant increases in total vBMD and bone strength parameters after sequential treatment with teriparatide followed by denosumab support the use of this regimen in PreMenIOP. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bone intrinsic material and compositional properties in postmenopausal women diagnosed with long-term Type-1 diabetes.

The incidence of diabetes mellitus and the associated complications are growing worldwide, affecting the patients' quality of life and exerting a considerable burden on health systems. Yet, the increase in fracture risk in type 1 diabetes (T1D) patients is not fully captured by bone mineral density (BMD), leading to the hypothesis that alterations in bone quality are responsible for the increased risk. Material/compositional properties are important aspects of bone quality, yet information on human bone material/compositional properties in T1D is rather sparse. The purpose of the present study is to measure both the intrinsic material behaviour by nanoindentation, and material compositional properties by Raman spectroscopy as a function of tissue age and microanatomical location (cement lines) in bone tissue from iliac crest biopsies from postmenopausal women diagnosed with long-term T1D (N = 8), and appropriate sex-, age-, BMD- and clinically-matched controls (postmenopausal women; N = 5). The results suggest elevation of advanced glycation endproducts (AGE) content in the T1D and show significant differences in mineral maturity / crystallinity (MMC) and glycosaminoglycan (GAG) content between the T1D and control groups. Furthermore, both hardness and modulus by nanoindentation are greater in T1D. These data suggest a significant deterioration of material strength properties (toughness) and compositional properties in T1D compared with controls.

Osteocyte lacunae in transiliac bone biopsy samples across life span.

Osteocytes act as bone mechanosensors, regulators of osteoblast/osteoclast activity and mineral homeostasis, however, knowledge about their functional/morphological changes throughout life is limited. We used quantitative backscattered electron imaging (qBEI) to investigate osteocyte lacunae sections (OLS) as a 2D-surrogate characterizing the osteocytes. OLS characteristics, the density of mineralized osteocyte lacunae (i.e., micropetrotic osteocytes, md.OLS-Density in nb/mm2) and the average degree of mineralization (CaMean in weight% calcium) of cortex and spongiosa were analyzed in transiliac biopsy samples from healthy individuals under 30 (n=59) and over 30 years (n=50) (i.e., before and after the age of peak bone mass, respectively). We found several differences in OLS-characteristics: 1). Inter-individually between the age groups: OLS-Density and OLS-Porosity were reduced by about 20% in older individuals in spongiosa and in cortex versus younger probands (both, p < 0.001). 2). Intra-individually between bone compartments: OLS-Density was higher in the cortex, +18.4%, p < 0.001 for younger and +7.6%, p < 0.05 for older individuals. Strikingly, the most frequent OLS nearest-neighbor distance was about 30 µm in both age groups and at both bone sites revealing a preferential organization of osteocytes in clusters. OLS-Density was negatively correlated with CaMean in both spongiosa and cortex (both, p < 0.001). Few mineralized OLS were found in young individuals along with an increase of md.OLS-Density with age. In summary, this transiliac bone sample analysis of 200000 OLS from 109 healthy individuals throughout lifespan reveals several age-related differences in OLS characteristics. Moreover, our study provides reference data from healthy individuals for different ages to be used for diagnosis of bone abnormalities in diseases. STATEMENT OF SIGNIFICANCE: Osteocytes are bone cells embedded in lacunae within the mineralized bone matrix and have a key role in the bone metabolism and the mineral homeostasis. Not easily accessible, we used quantitative backscattered electron imaging to determine precisely number and shape descriptors of the osteocyte lacunae in 2D. We analyzed transiliac biopsy samples from 109 individuals with age distributed from 2 to 95 years. Compact cortical bone showed constantly higher lacunar density than cancellous bone but the lacunar density in both bone tissue decreased with age before the peak bone mass age at 30 years and stabilized or even increased after this age. This extensive study provides osteocyte lacunae reference data from healthy individuals usable for bone pathology diagnosis.

Genome-wide association and replication studies identified TRHR as an important gene for lean body mass.

Low lean body mass (LBM) is related to a series of health problems, such as osteoporotic fracture and sarcopenia. Here we report a genome-wide association (GWA) study on LBM variation, by using Affymetrix 500K single-nucleotide polymorphism (SNP) arrays. In the GWA scan, we tested 379,319 eligible SNPs in 1,000 unrelated US whites and found that two SNPs, rs16892496 (p = 7.55 x 10(-8)) and rs7832552 (p = 7.58 x 10(-8)), within the thyrotropin-releasing hormone receptor (TRHR) gene were significantly associated with LBM. Subjects carrying unfavorable genotypes at rs16892496 and rs7832552 had, on average, 2.70 and 2.55 kg lower LBM, respectively, compared to those with alternative genotypes. We replicated the significant associations in three independent samples: (1) 1488 unrelated US whites, (2) 2955 Chinese unrelated subjects, and (3) 593 nuclear families comprising 1972 US whites. Meta-analyses of the GWA scan and the replication studies yielded p values of 5.53 x 10(-9) for rs16892496 and 3.88 x 10(-10) for rs7832552. In addition, we found significant interactions between rs16892496 and polymorphisms of several other genes involved in the hypothalamic-pituitary-thyroid and the growth hormone-insulin-like growth factor-I axes. Results of this study, together with the functional relevance of TRHR in muscle metabolism, support the TRHR gene as an important gene for LBM variation.

Genome-wide association and follow-up replication studies identified ADAMTS18 and TGFBR3 as bone mass candidate genes in different ethnic groups.

To identify and validate genes associated with bone mineral density (BMD), which is a prominent osteoporosis risk factor, we tested 379,319 SNPs in 1000 unrelated white U.S. subjects for associations with BMD. For replication, we genotyped the most significant SNPs in 593 white U.S. families (1972 subjects), a Chinese hip fracture (HF) sample (350 cases, 350 controls), a Chinese BMD sample (2955 subjects), and a Tobago cohort of African ancestry (908 males). Publicly available Framingham genome-wide association study (GWAS) data (2953 whites) were also used for in silico replication. The GWAS detected two BMD candidate genes, ADAMTS18 (ADAM metallopeptidase with thrombospondin type 1 motif, 18) and TGFBR3 (transforming growth factor, beta receptor III). Replication studies verified the significant findings by GWAS. We also detected significant associations with hip fracture for ADAMTS18 SNPs in the Chinese HF sample. Meta-analyses supported the significant associations of ADAMTS18 and TGFBR3 with BMD (p values: 2.56 x 10(-5) to 2.13 x 10(-8); total sample size: n = 5925 to 9828). Electrophoretic mobility shift assay suggested that the minor allele of one significant ADAMTS18 SNP might promote binding of the TEL2 factor, which may repress ADAMTS18 expression. The data from NCBI GEO expression profiles also showed that ADAMTS18 and TGFBR3 genes were differentially expressed in subjects with normal skeletal fracture versus subjects with nonunion skeletal fracture. Overall, the evidence supports that ADAMTS18 and TGFBR3 might underlie BMD determination in the major human ethnic groups.

Intrinsic material properties of trabecular bone by nanoindentation testing of biopsies taken from healthy women before and after menopause.

Postmenopausal osteoporosis in women is characterized by an increase in bone fragility and risk of fracture. In addition to transmenopausal decline in three-dimensional trabecular bone architecture, changes in intrinsic material properties (local stiffness, damping, and hardness) may contribute to increased bone fragility. In this study, nanoindentation was used to quantify transmenopausal changes in the intrinsic properties of trabecular bone. Paired transilial biopsy specimens were used from a previously reported study in which bone biopsies were obtained from women prior to menopause (premenopausal, age 49.0 ± 1.9) and at 12 months past the last menstrual period (postmenopausal, age 54.6 ± 2.2). Elastic and viscoelastic material properties of the trabecular bone were measured using quasi-static and dynamic nanoindentation techniques, respectively. Paired Student's t tests (n = 15) were performed to assess the significance of the measured intrinsic properties. Trabecular bone microarchitecture is compromised in postmenopausal women, and although this loss is associated with a trend toward reduction in some intrinsic properties (storage modulus), we found no statistically significant changes in bone intrinsic properties between healthy pre- and postmenopausal biopsies in the quasi-static results and frequency-averaged dynamic results.

Genome-wide association analyses identify SPOCK as a key novel gene underlying age at menarche.

For females, menarche is a most significant physiological event. Age at menarche (AAM) is a trait with high genetic determination and is associated with major complex diseases in women. However, specific genes for AAM variation are largely unknown. To identify genetic factors underlying AAM variation, a genome-wide association study (GWAS) examining about 380,000 SNPs was conducted in 477 Caucasian women. A follow-up replication study was performed to validate our major GWAS findings using two independent Caucasian cohorts with 854 siblings and 762 unrelated subjects, respectively, and one Chinese cohort of 1,387 unrelated subjects--all females. Our GWAS identified a novel gene, SPOCK (Sparc/Osteonectin, CWCV, and Kazal-like domains proteoglycan), which had seven SNPs associated with AAM with genome-wide false discovery rate (FDR) q<0.05. Six most significant SNPs of the gene were selected for validation in three independent replication cohorts. All of the six SNPs were replicated in at least one cohort. In particular, SNPs rs13357391 and rs1859345 were replicated both within and across different ethnic groups in all three cohorts, with p values of 5.09 x 10(-3) and 4.37 x 10(-3), respectively, in the Chinese cohort and combined p values (obtained by Fisher's method) of 5.19 x 10(-5) and 1.02 x 10(-4), respectively, in all three replication cohorts. Interestingly, SPOCK can inhibit activation of MMP-2 (matrix metalloproteinase-2), a key factor promoting endometrial menstrual breakdown and onset of menstrual bleeding. Our findings, together with the functional relevance, strongly supported that the SPOCK gene underlies variation of AAM.

Large-scale osteocyte lacunar morphological analysis of transiliac bone in normal and osteoporotic premenopausal women.

Bone's ability to adapt is governed by the network of embedded osteocytes, which inhabit individual pores called lacunae. The morphology of these lacunae and their resident osteocytes are known to change with age and diseases such as postmenopausal osteoporosis. However, it is unclear whether alterations in lacunar morphology are present in younger populations with osteoporosis. To investigate this, we implemented a previously validated methodology to image and quantify the three-dimensional morphometries of lacunae on a large scale with ultra-high-resolution micro-computed tomography (microCT) in transiliac bone biopsies from three groups of premenopausal women: control n = 39; idiopathic osteoporosis (IOP) n = 45; idiopathic low BMD (ILBMD) n = 19. Lacunar morphometric parameters were measured in both trabecular and cortical bone such as lacunar density (Lc.N/BV), lacunar volume (Lc.V), and lacunar sphericity (Lc.Sr). These were then compared against each other and also with previously measured tissue morphometries such as bone volume density (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and others. We detected no differences in lacunar morphology between the IOP, ILBMD and healthy premenopausal women. In contrast, we did find significant differences between lacunar morphologies including Lc.N/BV, Lc. V, and Lc. Sr in cortical and trabecular regions within all three groups (p < 0.001), which was consistent with our previous findings on a subgroup of the healthy group. Furthermore, we discovered strong correlations between Lc. Sr from trabecular regions with the measured BV/TV (R = -0.90, p < 0.05). The findings and comprehensive lacunar dataset we present here will be a crucial foundation for future investigations of the relationship between osteocyte lacunar morphology and disease.

A pilot study on the nanoscale properties of bone tissue near lacunae in fracturing women.

The goal of this study is to investigate the causes of osteoporosis-related skeletal fragility in postmenopausal women. We hypothesize that bone fragility in these individuals is largely due to mineral, and/or intrinsic material properties in the osteocyte lacunar/peri-lacunar regions of bone tissue. Innovative measurements with nanoscale resolution, including scanning electron microscope (SEM), an atomic force microscope that is integrated with infrared spectroscopy (AFM-IR), and nanoindentation, were used to characterize osteocyte lacunar and peri-lacunar properties in bone biopsies from fracturing (Cases) and matched (Age, BMD), non-fracturing (Controls) postmenopausal healthy women. In the peri-lacunar space, the nanoindentation results show that the modulus and hardness of the Controls are lower than the Cases. The AFM-IR results conclusively show that the mineral matrix, maturity (peak) (except in outer/far regions in Controls) were greater in Controls than in Cases. Furthermore, these results indicate that while mineral-to-matrix area ratio tend to be greater, the mineral maturity and crystallinity peak ratio "near" lacunae is greater than at regions "far" or more distance from lacunae in the Controls only. Due to the heterogeneity of bone structure, additional measurements are needed to provide more convincing evidence of altered lacunar characteristics and changes in the peri-lacunar bone as mechanisms related to postmenopausal women and fragility. Such findings would motivate new osteocyte-targeted treatments to reduce fragility fracture risks in these groups.

Denosumab After Teriparatide in Premenopausal Women With Idiopathic Osteoporosis.

CONTEXT: We have previously reported that teriparatide is associated with substantial increases in bone mineral density (BMD) at the lumbar spine (LS), total hip (TH), and femoral neck (FN) and small declines at the distal radius in 41 premenopausal women with idiopathic osteoporosis (IOP), all severely affected with low trauma fractures and/or very low BMD. Effects of teriparatide dissipate if not followed by antiresorptives. OBJECTIVE: To assess the effects of 12 and 24 months of denosumab in premenopausal women with IOP completing 24 months of teriparatide. METHODS: This was a preplanned phase 2B extension study. Premenopausal women with IOP who had completed a course of teriparatide received denosumab 60 mg every 6 months over 24 months. The main outcome measure was within-group change in BMD at the LS at 12 months. Secondary outcomes include change in 12-month BMD at other sites, 24-month BMD at all sites, trabecular bone score (TBS), and bone turnover markers (BTMs). RESULTS: After completing teriparatide, 32 participants took denosumab for 12 months and 29 for 24 months, with statistically significant increases in BMD at the LS (5.2 ± 2.6% and 6.9 ± 2.6%), TH (2.9 ± 2.4% and 4.6 ± 2.8%), and FN (3.0 ± 3.8% and 4.7 ± 4.9%). Over the entire 24-month teriparatide and 24-month denosumab treatment period, BMD increased by 21.9 ± 7.8% at the LS, 9.8 ± 4.6% at the TH, and 9.5 ± 4.7% at the FN (all P < .0001). TBS increased by 5.8 ± 5.6% (P < .001). Serum BTM decreased by 75% to 85% by 3 months and remained suppressed through 12 months of denosumab. Denosumab was generally well tolerated. CONCLUSION: These data support the use of sequential teriparatide and denosumab to increase BMD in premenopausal women with severe osteoporosis.

Spine Volumetric BMD and Strength in Premenopausal Idiopathic Osteoporosis: Effect of Teriparatide Followed by Denosumab.

CONTEXT: Premenopausal women with idiopathic osteoporosis (PreMenIOP) have marked deficits in bone density, microstructure, and strength. OBJECTIVE: To define effects of treatment with teriparatide followed by denosumab on lumbar spine (LS) volumetric bone mineral density (vBMD) and stiffness by finite element analysis assessed on central quantitative computed tomography (cQCT) scans. DESIGN, SETTINGS, AND PARTICIPANTS: Ancillary analysis of baseline, post-teriparatide, and post-denosumab cQCT scans from a randomized trial of 41 women allocated to teriparatide (20 mcg daily; n = 28) or placebo (n = 11). After 6 months, those on teriparatide continued for 18 months, and those on placebo switched to teriparatide for 24 months. After completing teriparatide, 33 enrolled in a Phase 2B extension with denosumab (60 mg every 6 months) for 12 months. MAIN OUTCOME MEASURES: Primary outcomes were percentage change from baseline in LS trabecular vBMD and stiffness after teriparatide and between end of teriparatide and completing denosumab. Percentage change from baseline in LS trabecular vBMD and stiffness after sequential teriparatide and denosumab were secondary outcomes. FINDINGS: There were large increases (all Ps < 0.001) in trabecular vBMD (25%), other vBMD parameters, and stiffness (21%) after teriparatide. Statistically significant increases in trabecular vBMD (10%; P < 0.001) and other vBMD parameters (P = 0.03-0.001) were seen after denosumab, while stiffness increased by 7% (P = 0.068). Sequential teriparatide and denosumab led to highly significant (all Ps < 0.001) increases LS trabecular vBMD (43%), other vBMD parameters (15-31%), and stiffness (21%). CONCLUSIONS: The large and statistically significant increases in volumetric density and stiffness after sequential treatment with teriparatide followed by denosumab are encouraging and support use of this regimen in PreMenIOP.

Whole exome sequencing reveals potentially pathogenic variants in a small subset of premenopausal women with idiopathic osteoporosis.

Osteoporosis in premenopausal women with intact gonadal function and no known secondary cause of bone loss is termed idiopathic osteoporosis (IOP). Women with IOP diagnosed in adulthood have profound bone structural deficits and often report adult and childhood fractures, and family history of osteoporosis. Some have very low bone formation rates (BFR/BS) suggesting osteoblast dysfunction. These features led us to investigate potential genetic etiologies of bone fragility. In 75 IOP women (aged 20-49) with low trauma fractures and/or very low BMD who had undergone transiliac bone biopsies, we performed Whole Exome Sequencing (WES) using our variant analysis pipeline to select candidate rare and novel variants likely to affect known disease genes. We ran rare-variant burden analyses on all genes individually and on phenotypically-relevant gene sets. For particular genes implicated in osteoporosis, we also assessed the frequency of all (including common) variants in subjects versus 6540 non-comorbid female controls. The variant analysis pipeline identified 4 women with 4 heterozygous variants in LRP5 and PLS3 that were considered to contribute to osteoporosis. All 4 women had adult fractures, and 3 women also had multiple fractures, childhood fractures and a family history of osteoporosis. Two women presented during pregnancy/lactation. In an additional 4 subjects, 4 different relevant Variants of Uncertain Significance (VUS) were detected in the genes FKBP10, SLC34A3, and HGD. Of the subjects with VUS, 2 had multiple adult fractures, childhood fractures, and presented during pregnancy/lactation, and 2 had nephrolithiasis. BFR/BS varied among the 8 subjects with identified variants; BFR/BS was quite low in those with variants that are likely to have adverse effects on bone formation. The analysis pipeline did not discover candidate variants in COL1A1, COL1A2, WNT, or ALPL. Although we found several novel and rare variants in LRP5, cases did not have an increased burden of common LRP5 variants compared to controls. Cohort-wide collapsing analysis did not reveal any novel disease genes with genome-wide significance for qualifying variants between controls and our 75 cases. In summary, WES revealed likely pathogenic variants or relevant VUS in 8 (11%) of 75 women with IOP. Notably, the genetic variants identified were consistent with the affected women's diagnostic evaluations that revealed histological evidence of low BFR/BS or biochemical evidence of increased bone resorption and urinary calcium excretion. These results, and the fact that the majority of the women had no identifiable genetic etiology, also suggest that the pathogenesis of and mechanisms leading to osteoporosis in this cohort are heterogeneous. Future research is necessary to identify both new genetic and non-genetic etiologies of early-onset osteoporosis.

Genome-wide association study identifies two novel loci containing FLNB and SBF2 genes underlying stature variation.

Human stature, as an important physical index in clinical practice and a usual covariate in gene mapping of complex disorders, is a highly heritable complex trait. To identify specific genes underlying stature, a genome-wide association study was performed in 1000 unrelated homogeneous Caucasian subjects using Affymetrix 500K arrays. A group of seven contiguous markers in the region of SBF2 gene (Set-binding factor 2) are associated with stature, significantly so at the genome-wide level after false discovery rate (FDR) correction (FDR q = 0.034-0.042). Three SNPs in another SNP group in the Filamin B (FLNB) gene were also associated with stature, significantly so with FDR q = 0.042-0.048. In follow-up independent replication studies, rs10734652 in the SBF2 gene was significantly (P = 0.036) and suggestively (P = 0.07) associated with stature in Caucasian families and 1306 unrelated Caucasian subjects, respectively, and rs9834312 in the FLNB gene was also associated with stature in such two independent Caucasian populations (P = 0.008 in unrelated sample and P = 0.049 in family sample). Particularly, additional significant replication association signals were detected in Chinese, an ethnic population different from Caucasian, between rs9834312 and stature in 619 unrelated northern Chinese subjects (P = 0.017), as well as between rs10734652 and stature in 2953 unrelated southern Chinese subjects (P = 0.048). This study also provides additional replication evidence for some of the already published stature loci. These results, together with the known functional relevance of the SBF2 and FLNB genes to skeletal linear growth and bone formation, support that two regions containing FLNB and SBF2 genes are two novel loci underlying stature variation.

Genome-wide copy-number-variation study identified a susceptibility gene, UGT2B17, for osteoporosis.

Osteoporosis, a highly heritable disease, is characterized mainly by low bone-mineral density (BMD), poor bone geometry, and/or osteoporotic fractures (OF). Copy-number variation (CNV) has been shown to be associated with complex human diseases. The contribution of CNV to osteoporosis has not been determined yet. We conducted case-control genome-wide CNV analyses, using the Affymetrix 500K Array Set, in 700 elderly Chinese individuals comprising 350 cases with homogeneous hip OF and 350 matched controls. We constructed a genomic map containing 727 CNV regions in Chinese individuals. We found that CNV 4q13.2 was strongly associated with OF (p = 2.0 x 10(-4), Bonferroni-corrected p = 0.02, odds ratio = 1.73). Validation experiments using PCR and electrophoresis, as well as real-time PCR, further identified a deletion variant of UGT2B17 in CNV 4q13.2. Importantly, the association between CNV of UGT2B17 and OF was successfully replicated in an independent Chinese sample containing 399 cases with hip OF and 400 controls. We further examined this CNV's relevance to major risk factors for OF (i.e., hip BMD and femoral-neck bone geometry) in both Chinese (689 subjects) and white (1000 subjects) samples and found consistently significant results (p = 5.0 x 10(-4) -0.021). Because UGT2B17 encodes an enzyme catabolizing steroid hormones, we measured the concentrations of serum testosterone and estradiol for 236 young Chinese males and assessed their UGT2B17 copy number. Subjects without UGT2B17 had significantly higher concentrations of testosterone and estradiol. Our findings suggest the important contribution of CNV of UGT2B17 to the pathogenesis of osteoporosis.

let-7 microRNAs induce tamoxifen sensitivity by downregulation of estrogen receptor α signaling in breast cancer.

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3'UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3'UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.