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.

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.

Titin mutation in familial restrictive cardiomyopathy.

BACKGROUND: Familial restrictive cardiomyopathy (RCM) caused by a single gene mutation is the least common of the inherited cardiomyopathies. Only a few RCM-causing mutations have been described. Most mutations causing RCM are located in sarcomere protein genes which also cause hypertrophic cardiomyopathy (HCM). Other genes associated with RCM include the desmin and familial amyloidosis genes. In the present study we describe familial RCM with severe heart failure triggered by a de novo mutation in TTN, encoding the huge muscle filament protein titin. METHODS AND RESULTS: Family members underwent physical examination, ECG and Doppler echocardiogram studies. The family comprised 6 affected individuals aged 12-35 years. Linkage to candidate loci was performed, followed by gene sequencing. Candidate loci/gene analysis excluded 18 candidate genes but showed segregation with a common haplotype surrounding the TTN locus. Sequence analysis identified a de novo mutation within exon 266 of the TTN gene, resulting in the replacement of tyrosine by cysteine. p.Y7621C affects a highly conserved region in the protein within a fibronectin-3 domain, belonging to the A/I junction region of titin. No other disease-causing mutation was identified in cardiomyopathy genes by whole exome sequencing. CONCLUSIONS: Our study shows, for the first time, that mutations in TTN can cause restrictive cardiomyopathy. The giant filament titin is considered to be a determinant of a resting tension of the sarcomere and this report provides genetic evidence of its crucial role in diastolic function.

Analyses of RANK and RANKL in the post-GWAS context: functional evidence of vitamin D stimulation through a RANKL distal region.

Over the past decade, many genome-wide association studies (GWAs) and meta-analyses have identified genes and regions involved in osteoporotic phenotypes. Nevertheless, the large majority of these results were not tested at any functional level. GWA-associated single-nucleotide polymorphisms (SNPs) near candidate genes such as RANK and RANKL suggest that these SNPs and/or other variants nearby may be involved in bone phenotype determination. This study focuses on SNPs along these two genes, which encode proteins with a well-established role in the bone remodeling equilibrium. Thirty-three SNPs, chosen for their location in evolutionary conserved regions or replicated from previous studies, were genotyped in the BARCOS cohort of 1061 postmenopausal women and tested for association with osteoporotic phenotypes. SNP rs9594738, which lies 184 kb upstream of the RANKL gene, was the only SNP found to be associated with a bone phenotype (dominant model: beta coefficient = -0.034, p = 1.5 × 10(-4) , for lumbar spine bone mineral density). Functional experiments exploring a distal region (DR) of 831 bp that harbors this SNP in a centered position (nt 470) demonstrated its capacity to inhibit the RANKL promoter in reporter gene assays. Remarkably, this DR inhibition was significantly reduced in the presence of vitamin D. In conclusion, the GWA-associated SNP rs9594738 lies in a region involved in transcription regulation through which vitamin D could be regulating RANKL expression and bone mineral density.

Microindentation for in vivo measurement of bone tissue material properties in atypical femoral fracture patients and controls.

Atypical femoral fractures (AFF) associated with long-term bisphosphonates (LTB) are a growing concern. Their etiology is unknown, but bone material properties might be deteriorated. In an AFF series, we analyzed the bone material properties by microindentation. Four groups of patients were included: 6 AFF, 38 typical osteoporotic fractures, 6 LTB, and 20 controls without fracture. Neither typical osteoporotic fractures nor controls have received any antiosteoporotic medication. A general laboratory workup, bone densitometry by dual-energy X-ray absorptiometry (DXA), and microindentation testing at the tibia were done in all patients. Total indentation distance (Total ID), indentation distance increase (IDI), and creep indentation distance (Creep ID) were measured (microns). Age-adjusted analysis of covariance (ANCOVA) was used for comparisons. Controls were significantly younger than fracture groups. Bisphosphonate exposure was on average 5.5 years (range 5 to 12 years) for the AFF and 5.4 years (range 5 to 8 years) for the LTB groups. Total ID (microns) showed better material properties (lower Total ID) for controls 36 (± 6; mean ± SD) than for AFF 46 (± 4) and for typical femoral fractures 47 (± 13), respectively. Patients on LTB showed values between controls and fractures, 38 (± 4), although not significantly different from any of the other three groups. IDI values showed a similar pattern 13 (± 2), 16 (± 6), 19 (± 3), and 18 (± 5). After adjusting by age, significant differences were seen between controls and typical (p < 0.001) and atypical fractures (p = 0.03) for Total ID and for IDI (p < 0.001 and p < 0.05, respectively). There were no differences in Creep ID between groups. Our data suggest that patients with AFF have a deep deterioration in bone material properties at a tissue level similar to that for the osteoporotic fracture group. The LTB group shows levels that are in between controls and both type of fractures, although not statistically different. These results suggest that bisphosphonate therapy probably does not put the majority of patients at risk for AFF.

A novel titin mutation in adult-onset familial dilated cardiomyopathy.

Familial dilated cardiomyopathy is a major cause of advanced heart failure and heart transplantation. In most families, the disease-causing mutation is unknown, and relatives should therefore undergo periodic screening to facilitate early diagnosis and therapy. In the present study, we describe a novel titin truncation mutation causing adult-onset familial dilated cardiomyopathy in an Israeli Arab family. The family members underwent physical examination, electrocardiography, and Doppler echocardiography. Linkage to candidate loci was performed, followed by gene sequencing. We identified 13 clinically affected family members (8 men and 5 women, mean age 47 ± 12 years). Compared with their healthy first-degree relatives, the affected relatives had a larger end-diastolic left ventricular dimension (60 ± 10 vs 49 ± 4 mm, p <0.001), lower ejection fraction (43 ± 11% vs 60 ± 6%, p <0.001), and markedly higher end-systolic volume indexes but no difference in wall thickness or diastolic function. The linkage studies or direct sequencing excluded LMNA, MYH7, TNNT2, TNNI3, SCN5A, DES, SGCD, ACTC, PLN, and MYH6 but established linkage to the TTN locus at chromosome 2q31, yielding a maximum (2-point) LOD score of 3.44. Sequence analysis identified an insertion (c.58880insA), causing protein truncation after 19,628 amino acids (p.S19628IfsX1). No founder effect was found among the Israeli Arabs. In conclusion, titin is a giant protein with a key role in sarcomere assembly, force transmission, and maintenance of resting tension. Although some mutations result in skeletal myopathy, others cause isolated, maturity-onset cardiomyopathy.

COL1A1 haplotypes and hip fracture.

Fragility fractures resulting from low-trauma events such as a fall from standing height are associated with osteoporosis and are very common in older people, especially women. Three single nucleotide polymorphisms (SNPs) at the COL1A1 gene (rs1107946, rs11327935, and rs1800012) have been widely studied and previously associated with bone mineral density (BMD) and fracture. A rare haplotype (T-delT-T) of these three SNPs was found to be greatly overrepresented in fractured individuals compared with nonfractured controls, thus becoming a good candidate for predicting increased fracture risk. The aim of our study was to assess the association of this haplotype with fracture risk in Spanish individuals. We recruited two independent groups of ∼100 patients with hip fracture (a total of 203 individuals) and compared the genotype and haplotype distributions of the three SNPs in the fractured patients with those of 397 control individuals from the BARCOS Spanish cohort. We found no association with risk of fracture at the genotype level for any of the SNPs, and no differences in the SNP frequencies between the two groups. At the haplotype level, we found no association between the T-delT-T haplotype and fracture. However, we observed a small but significant (p = 0.03) association with another rare haplotype, G-insT-T, which was slightly overrepresented in the patient group.

Functional relevance of the BMD-associated polymorphism rs312009: novel involvement of RUNX2 in LRP5 transcriptional regulation.

LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway.

Effect of IL-1beta, PGE(2), and TGF-beta1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts.

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)-1beta, proftaglandin E(2) (PGE(2)), and transforming growth factor-beta1 (TGF-beta1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real-time PCR. In all cultures, OPG mRNA increased significantly in response to IL-1beta treatment and decreased in response to TGF-beta1 whereas PGE(2) treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL-1beta treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF-beta1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption.

A combined approach to the molecular analysis of cystinuria: from urinalysis to sequencing via genotyping.

BACKGROUND: Cystinuria is an autosomal recessive disease that is manifested by kidney stones and is caused by mutations in two genes: SLC3AI on chromosome 2p and SLC7A9 on chromosome 19q. Urinary cystine levels in obligate carriers are often, but not always, helpful in identifying the causative gene. OBJECTIVES: To characterize the clinical features and analyze the genetic basis of cystinuria in an inbred Moslem Arab Israeli family. METHODS: Family members were evaluated for urinary cystine and amino acid levels. DNA was initially analyzed with polymorphic markers close to the two genes and SLC7A9 was fully sequenced. RESULTS: Full segregation was found with the marker close to SLC7A9. Sequencing of this gene revealed a missense mutation, P482L, in the homozygous state in all three affected sibs. CONCLUSIONS: A combination of urinary cystine levels in obligate carriers, segregation analysis with polymorphic markers, and sequencing can save time and resources in the search for cystinuria mutations.