Metals accumulation affects bone and muscle in osteoporotic patients: A pilot study.

Osteoporosis is the most common bone disease, characterized by decreased bone mineral density (BMD) and often associated to decreased muscle mass and function. Metal exposure plays a role in the pathophysiology of osteoporosis and affects also muscle quality. The aim of this study was to assess the association between metal levels in bone and muscle samples and the degeneration of these tissues. A total of 58 subjects (30 male and 28 female) was enrolled and classified in osteoporotic (OP, n = 8), osteopenic (Ope, n = 30) and healthy (CTR, n = 20) subjects, according to BMD measures. Femoral head bone samples and vastus lateralis muscle samples were collected during hip arthroplasty surgeries. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis showed increased levels of Al, Cd and Pb in OP and Ope bone tissue compared to CTR subjects (p = 0.04, p = 0.005 and p = 0.01, respectively). Whereas, increased levels of Co, Cd and Pb were measured in OP and Ope muscle tissues, compared to CTRs (p < 0.001, p = 0.02 and p = 0.01, respectively). In addition, Al, Cd and Pb levels in bone and Cd and Co levels in muscle were negatively correlated with BMD. A negative association among Co, Cd, Cr and Hg levels and muscle fibers diameter was also observed in muscle tissues. This study assessed that metal exposure can affects bone and muscle tissue quality and may contribute to the onset and progression of musculoskeletal diseases such as osteoporosis. Therefore, it is important to implement metal exposure assessment and their impact on disease development, in order to manage and prevent metal accumulation effects on bone and muscle quality.

In Cis Effect of DMPK Expanded Alleles in Myotonic Dystrophy Type 1 Patients Carrying Variant Repeats at 5' and 3' Ends of the CTG Array.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disease caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK gene. DM1 alleles containing non-CTG variant repeats (VRs) have been described, with uncertain molecular and clinical consequences. The expanded trinucleotide array is flanked by two CpG islands, and the presence of VRs could confer an additional level of epigenetic variability. This study aims to investigate the association between VR-containing DMPK alleles, parental inheritance and methylation pattern of the DM1 locus. The DM1 mutation has been characterized in 20 patients using a combination of SR-PCR, TP-PCR, modified TP-PCR and LR-PCR. Non-CTG motifs have been confirmed by Sanger sequencing. The methylation pattern of the DM1 locus was determined by bisulfite pyrosequencing. We characterized 7 patients with VRs within the CTG tract at 5' end and 13 patients carrying non-CTG sequences at 3' end of the DM1 expansion. DMPK alleles with VRs at 5' end or 3' end were invariably unmethylated upstream of the CTG expansion. Interestingly, DM1 patients with VRs at the 3' end showed higher methylation levels in the downstream island of the CTG repeat tract, preferentially when the disease allele was maternally inherited. Our results suggest a potential correlation between VRs, parental origin of the mutation and methylation pattern of the DMPK expanded alleles. A differential CpG methylation status could play a role in the phenotypic variability of DM1 patients, representing a potentially useful diagnostic tool.

Role of the Vitamin D Receptor (VDR) in the Pathogenesis of Osteoporosis: A Genetic, Epigenetic and Molecular Pilot Study.

The vitamin D receptor (VDR) regulates bone development and calcium homeostasis, suggesting a central role in musculoskeletal diseases such as osteoporosis (OP). Several studies have examined the contribution of VDR polymorphisms and epigenetic signatures in bone metabolism and OP risk, with sometimes inconclusive results. Our study aimed to explore the association between genetic variability, expression and the methylation pattern of VDR with the risk of OP in a cohort of Caucasian patients. Genomic DNA from 139 OP, 54 osteopenic (Ope) and 73 healthy (CTR) subjects were used for genotyping the rs731236 (TaqI), rs2228570 (FokI) and rs11568820 (Cdx2) polymorphisms of the VDR gene by an allelic discrimination assay. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of VDR expression levels and pyrosequencing analysis of a VDR promoter CpG island were carried out in a subcohort (25 OP and 25 CTR) of subjects. Data obtained showed a significantly higher OP risk for rs11568820 G/A and A/A genotypes (p = 0.05). qRT-PCR revealed lower VDR gene expression levels in the OP group compared to CTR subjects (p = 0.0009), also associated with both the rs11568820 A/A genotype (p = 0.03) and femoral fragility fractures (p = 0.05). No association was found between the methylation pattern of the region analyzed of the VDR promoter and its expression levels. Our results identify a significative association between Cdx2 rs11568820 polymorphism and OP risk. In addition, the VDR transcriptomic profile suggests a putative interconnection with OP progression, providing a useful tool to stratify OP phenotype and fragility fracture risk.

Methylation of the Vitamin D Receptor Gene in Human Disorders.

The Vitamin D Receptor (VDR) mediates the actions of 1,25-Dihydroxvitamin D3 (1,25(OH)2D3), which has important roles in bone homeostasis, growth/differentiation of cells, immune functions, and reduction of inflammation. Emerging evidences suggest that epigenetic modifications of the VDR gene, particularly DNA methylation, may contribute to the onset and progression of many human disorders. This review aims to summarize the available information on the role of VDR methylation signatures in different pathological contexts, including autoimmune diseases, infectious diseases, cancer, and others. The reversible nature of DNA methylation could enable the development of therapeutic strategies, offering new avenues for the management of these worldwide diseases.

Sarcopenia and bone health: new acquisitions for a firm liaison.

Osteosarcopenia (OS) is a newly defined condition represented by the simultaneous presence of osteopenia/osteoporosis and sarcopenia, the main age-related diseases. The simultaneous coexistence of the two phenotypes derives from the close connection of the main target tissues involved in their pathogenesis: bone and muscle. These two actors constitute the bone-muscle unit, which communicates through a biochemical and mechanical crosstalk which involves multiple factors. Altered pattern of molecular pathways leads to an impairment of both the functionality of the tissue itself and the communication with the complementary tissue, composing the OS pathogenesis. Recent advances in the genetics field have provided the opportunity to delve deeper into the complex biological and molecular mechanisms underlying OS. Unfortunately, there are still many gaps in our understanding of these pathways, but it has proven essential to apply strategies such as exercise and nutritional intervention to counteract OS. New therapeutic strategies that simultaneously target bone and muscle tissue are limited, but recently new targets for the development of dual-action drug therapies have been identified. This narrative review aims to provide an overview of the latest scientific evidence associated with OS, a complex disorder that will pave the way for future research aimed at understanding the bone-muscle-associated pathogenetic mechanisms.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.

Deregulated Clusterin as a Marker of Bone Fragility: New Insights into the Pathophysiology of Osteoporosis.

Clusterin (CLU) is a secreted heterodimeric glycoprotein expressed in all organism fluids as well as in the intracellular matrix that plays key roles in several pathological processes. Its recent involvement in muscle degeneration of osteoporotic patients led to investigation of the role of CLU in bone metabolism, given the biochemical and biomechanical crosstalk of the bone-muscle unit. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis of CLU expression was performed in both osteoblasts and Peripheral Blood Mononuclear Cells (PBMCs) from osteoporotic patients (OP) and healthy individuals (CTR). Furthermore, immunohistochemical analysis on femoral head tissues and enzyme-linked immunosorbent assay (ELISA) in plasma samples were performed to investigate CLU expression pattern. Finally, genotyping of CLU rs11136000 polymorphism has also been performed by qRT-PCR assays to explore a possible association with CLU expression levels. Data obtained showed a significantly increased expression level of secreted CLU isoform in PBMCs and osteoblasts from OP patients. Immunohistochemical analysis confirms the increased expression of CLU in OP patients, both in osteocytes and osteoblasts, while plasma analysis reveals a statistically significant decrease of CLU levels. Unfortunately, no functional association between CLU expression levels and the presence of CLU rs11136000 polymorphism in OP patients was found. These data suggest a potential role played by CLU as a potential biomarker for the diagnosis and prognosis of OP progression.

Epigenetics of Myotonic Dystrophies: A Minireview.

Myotonic dystrophy type 1 and 2 (DM1 and DM2) are two multisystemic autosomal dominant disorders with clinical and genetic similarities. The prevailing paradigm for DMs is that they are mediated by an in trans toxic RNA mechanism, triggered by untranslated CTG and CCTG repeat expansions in the DMPK and CNBP genes for DM1 and DM2, respectively. Nevertheless, increasing evidences suggest that epigenetics can also play a role in the pathogenesis of both diseases. In this review, we discuss the available information on epigenetic mechanisms that could contribute to the DMs outcome and progression. Changes in DNA cytosine methylation, chromatin remodeling and expression of regulatory noncoding RNAs are described, with the intent of depicting an epigenetic signature of DMs. Epigenetic biomarkers have a strong potential for clinical application since they could be used as targets for therapeutic interventions avoiding changes in DNA sequences. Moreover, understanding their clinical significance may serve as a diagnostic indicator in genetic counselling in order to improve genotype-phenotype correlations in DM patients.

T-Score and Handgrip Strength Association for the Diagnosis of Osteosarcopenia: A Systematic Review and Meta-Analysis.

BACKGROUND: Osteosarcopenia is a recently identified condition caused by the coexistence of osteoporosis and sarcopenia that affects the frail elderly population, leading to an increased risk of falls and fractures. Given the recent socio-economic interest associated with osteosarcopenia, the aim of this meta-analysis is to provide an overview of the factors potentially involved in its pathogenesis, assessing its population type, prevalence, and associated variables. METHODS: A comprehensive systematic search for relevant studies, published from 2015 to 2020, was performed by using PubMed, EMBASE, and Cochrane databases. We analysed the variables of age, vitamin D, handgrip, and T-score in four different groups: healthy, osteopenic-osteoporotic, sarcopenic, and osteosarcopenic. RESULTS: A total of 6504 patients from 16 studies were included in the final meta-analysis. The analysis of the individual variables reveals a statistically significant correlation between the handgrip test data and T-score (p < 0.001). CONCLUSIONS: The correlation between T-score values and handgrip strength suggests a new potential parameter in the development of predictive models that could be used in clinical practice, highlighting its importance for the diagnosis of osteosarcopenia.

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

The long pentraxin PTX3: a novel serum marker to improve the prediction of osteoporosis and osteoarthritis bone-related phenotypes.

BACKGROUND: The long pentraxin PTX3 is generating great interest given the recent discovery of its involvement in bone metabolism. This study investigates the role of circulating PTX3 as a marker of bone-related phenotypes in patients with osteoporosis (OP) and osteoarthritis (OA). METHODS: Serum PTX3 levels were determined using an enzyme-linked immunosorbent assay (ELISA) in a total of OP (n=32), OA (n=19) patients and healthy controls (CTR; n=25). ROC curve analysis was carried out to evaluate the potential of PTX3 for the diagnosis of bone-related phenotypes. In addition, the association between PTX3 serum levels and biochemical markers was estimated by Spearman correlation analysis. RESULTS: Serum analysis reveals a statistically significant increase of PTX3 levels in OP and OA patients, compared to CTR subjects (**** p < 0.0001, **** p < 0.0001). ROC curve of PTX3 levels exhibits an excellent sensitivity and specificity for OP and OA diseases (**** p < 0.0001 and **** p < 0.0001, respectively). Moreover, serum PTX3 levels are positively associated with ALP (r = - 0.5257, p = 0.0083) and PTH levels (r = 0.4704, p = 0.0203) in OP patients. CONCLUSIONS: These results confirm the pivotal role of PTX3 in bone metabolism and suggest its potential use as a predictor of OP and OA bone-related phenotypes.

DNA Methylation Signatures of Bone Metabolism in Osteoporosis and Osteoarthritis Aging-Related Diseases: An Updated Review.

DNA methylation is one of the most studied epigenetic mechanisms that play a pivotal role in regulating gene expression. The epigenetic component is strongly involved in aging-bone diseases, such as osteoporosis and osteoarthritis. Both are complex multi-factorial late-onset disorders that represent a globally widespread health problem, highlighting a crucial point of investigations in many scientific studies. In recent years, new findings on the role of DNA methylation in the pathogenesis of aging-bone diseases have emerged. The aim of this systematic review is to update knowledge in the field of DNA methylation associated with osteoporosis and osteoarthritis, focusing on the specific tissues involved in both pathological conditions.

Genetic variability in noncoding RNAs: involvement of miRNAs and long noncoding RNAs in osteoporosis pathogenesis.

The pathogenesis of osteoporosis is multifactorial and is the consequence of genetic, hormonal and lifestyle factors. Epigenetics, including noncoding RNA (ncRNA) deregulation, represents a link between susceptibility to develop the disease and environmental influences. The majority of studies investigated the expression of ncRNAs in osteoporosis patients; however, very little information is available on their genetic variability. In this review, we focus on two classes of ncRNAs: miRNAs and long noncoding RNAs (lncRNAs). We summarize recent findings on how polymorphisms in miRNAs and lncRNAs can perturb the lncRNA/miRNA/mRNA axis and may be involved in osteoporosis clinical outcome. We also provide a general overview on databases and bioinformatic tools useful for associating miRNAs and lncRNAs variability with complex genetic diseases.

Circulating Long Non-Coding RNA GAS5 Is Overexpressed in Serum from Osteoporotic Patients and Is Associated with Increased Risk of Bone Fragility.

Osteoporosis (OP) is a multifactorial disorder in which environmental factors along with genetic variants and epigenetic mechanisms have been implicated. Long non-coding RNAs (lncRNAs) have recently emerged as important regulators of bone metabolism and OP aetiology. In this study, we analyzed the expression level and the genetic association of lncRNA GAS5 in OP patients compared to controls. Quantitative RT-PCR analysis of GAS5 was performed on the serum of 56 OP patients and 28 healthy individuals. OP subjects were divided into three groups of analysis: 29 with fragility fractures of lumbar spine (OP_VF), 14 with fragility fractures of femoral neck (OP_FF) and 13 without fractures (OP_WF). Genotyping of the rs145204276 insertion/deletion polymorphism has also been performed by Restriction fragment length polymorphism (RFLP) and direct sequencing analyses. Expression of circulating GAS5 is significantly increased in OP patients compared to controls (p < 0.01), with a statistically higher significance in fractured OP individuals vs. healthy subjects (p < 0.001). No statistically significant change was found in female OP patients; conversely, GAS5 is upregulated in the subgroup of fractured OP women sera (p < 0.01) and in all OP males (p < 0.05). Furthermore, a direct correlation between GAS5 expression level and parathyroid hormone (PTH) concentration was found in OP patients (r = 0.2930; p = 0.0389). Genetic analysis of rs145204276 revealed that the deletion allele was correlated with a higher expression of GAS5 in OP patients (0.22 ± 0.02 vs. 0.15 ± 0.01, ** p < 0.01). Our results suggest circulating GAS5 as a putative biomarker for the diagnosis and prognosis of OP and OP-related fractures.

Identification of Aberrantly-Expressed Long Non-Coding RNAs in Osteoblastic Cells from Osteoporotic Patients.

Osteoporosis (OP) is a multifactorial disease influenced by genetic, epigenetic, and environmental factors. One of the main causes of the bone homeostasis alteration is inflammation resulting in excessive bone resorption. Long non-coding RNAs (lncRNAs), have a crucial role in regulating many important biological processes in bone, including inflammation. We designed our study to identify lncRNAs misregulated in osteoblast primary cultures derived from OP patients (n = 4), and controls (CTRs, n = 4) with the aim of predicting possible RNA and/or protein targets implicated in this multifactorial disease. We focused on 84 lncRNAs regulating the expression of pro-inflammatory and anti-inflammatory genes and miRNAs. In silico analysis was utilized to predict the interaction of lncRNAs with miRNAs, mRNAs, and proteins targets. Six lncRNAs were significantly down-regulated in OP patients compared to controls: CEP83-AS1, RP11-84C13.1, CTC-487M23.5, GAS5, NCBP2-AS2, and SDCBP2-AS1. Bioinformatic analyses identified HDCA2, PTX3, and FGF2 proteins as downstream targets of CTC-487M23.5, GAS5, and RP11-84C13.1 lncRNAs mediated by the interaction with miRNAs implicated in OP pathogenesis, including miR-21-5p. Altogether, these data open a new regulatory mechanism of gene expression in bone homeostasis and could direct the development of future therapeutic approaches.

The Role of PTX3 in Mineralization Processes and Aging-Related Bone Diseases.

The Long Pentraxin 3 (PTX3) is a multifunctional glycoprotein released by peripheral blood leukocytes and myeloid dendritic cells in response to primary pro-inflammatory stimuli, that acts as a non-redundant component of the humoral arm of innate immunity. In addition to the primary role in the acute inflammatory response, PTX3 seems to be involved in other physiological and pathological processes. Indeed, PTX3 seems to play a pivotal role in the deposition and remodeling of bone matrix during the mineralization process, promoting osteoblasts differentiation and activity. Recently, PTX3 was seen to be involved in the ectopic calcifications' formation in breast cancer disease. In this regard, it has been observed that breast cancer tumors characterized by high expression of PTX3 and high amount of Breast Osteoblast Like Cells (BOLCs) showed several Hydroxyapatite (HA) microcalcifications, suggesting a likely role for PTX3 in differentiation and osteoblastic activity in both bone and extra-bone sites. Furthermore, given its involvement in bone metabolism, several studies agree with the definition of PTX3 as a molecule significantly involved in the pathogenesis of age-related bone diseases, such as osteoporosis, both in mice and humans. Recent results suggest that genetic and epigenetic mechanisms acting on PTX3 gene are also involved in the progression of these diseases. Based on these evidences, the aim of our systemic review was to offer an overview of the variety of biological processes in which PTX3 is involved, focusing on bone mineralization, both in a physiological and pathological context.