Publications by authors named "Kaare M Gautvik"

38 Publications

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture, Studied in Weight-Bearing and Non-Weight-Bearing Human Bone.

J Bone Miner Res 2020 06 2;35(6):1065-1076. Epub 2020 Mar 2.

Unger-Vetlesen Institute, Lovisenberg Diaconal Hospital, Oslo, Norway.

We investigated mechanisms resulting in low bone mineral density (BMD) and susceptibility to fracture by comparing noncoding RNAs (ncRNAs) in biopsies of non-weight-bearing (NWB) iliac (n = 84) and weight bearing (WB) femoral (n = 18) postmenopausal bone across BMDs varying from normal (T-score > -1.0) to osteoporotic (T-score ≤ -2.5). Global bone ncRNA concentrations were determined by PCR and microchip analyses. Association with BMD or fracture, adjusted by age and body mass index, were calculated using linear and logistic regression and least absolute shrinkage and selection operator (Lasso) analysis. At 10% false discovery rate (FDR), 75 iliac bone ncRNAs and 94 femoral bone ncRNAs were associated with total hip BMD. Eight of the ncRNAs were common for the two sites, but five of them (miR-484, miR-328-3p, miR-27a-5p, miR-28-3p, and miR-409-3p) correlated positively to BMD in femoral bone, but negatively in iliac bone. Of predicted pathways recognized in bone metabolism, ECM-receptor interaction and proteoglycans in cancer emerged at both sites, whereas fatty acid metabolism and focal adhesion were only identified in iliac bone. Lasso analysis and cross-validations identified sets of nine bone ncRNAs correlating strongly with adjusted total hip BMD in both femoral and iliac bone. Twenty-eight iliac ncRNAs were associated with risk of fracture (FDR < 0.1). The small nucleolar RNAs, RNU44 and RNU48, have a function in stabilization of ribosomal RNAs (rRNAs), and their association with fracture and BMD suggest that aberrant processing of rRNAs may be involved in development of osteoporosis. Cis-eQTL (expressed quantitative trait loci) analysis of the iliac bone biopsies identified two loci associated with microRNAs (miRNAs), one previously identified in a heel-BMD genomewide association study (GWAS). In this comprehensive investigation of the skeletal genetic background in postmenopausal women, we identified functional bone ncRNAs associated to fracture and BMD, representing distinct subsets in WB and NWB skeletal sites. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3974DOI Listing
June 2020

Mendelian Randomization Analysis Reveals a Causal Influence of Circulating Sclerostin Levels on Bone Mineral Density and Fractures.

J Bone Miner Res 2019 10 2;34(10):1824-1836. Epub 2019 Aug 2.

MRC Integrative Epidemiology Unit (IEU), Bristol Medical School, University of Bristol, Bristol, UK.

In bone, sclerostin is mainly osteocyte-derived and plays an important local role in adaptive responses to mechanical loading. Whether circulating levels of sclerostin also play a functional role is currently unclear, which we aimed to examine by two-sample Mendelian randomization (MR). A genetic instrument for circulating sclerostin, derived from a genomewide association study (GWAS) meta-analysis of serum sclerostin in 10,584 European-descent individuals, was examined in relation to femoral neck bone mineral density (BMD; n = 32,744) in GEFOS and estimated bone mineral density (eBMD) by heel ultrasound (n = 426,824) and fracture risk (n = 426,795) in UK Biobank. Our GWAS identified two novel serum sclerostin loci, B4GALNT3 (standard deviation [SD]) change in sclerostin per A allele (β = 0.20, p = 4.6 × 10 ) and GALNT1 (β  = 0.11 per G allele, p = 4.4 × 10 ). B4GALNT3 is an N-acetyl-galactosaminyltransferase, adding a terminal LacdiNAc disaccharide to target glycocoproteins, found to be predominantly expressed in kidney, whereas GALNT1 is an enzyme causing mucin-type O-linked glycosylation. Using these two single-nucleotide polymorphisms (SNPs) as genetic instruments, MR revealed an inverse causal relationship between serum sclerostin and femoral neck BMD (β = -0.12, 95% confidence interval [CI] -0.20 to -0.05) and eBMD (β = -0.12, 95% CI -0.14 to -0.10), and a positive relationship with fracture risk (β = 0.11, 95% CI 0.01 to 0.21). Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower eBMD, and greater B4GALNT3 expression in arterial tissue (probability >99%). Our findings suggest that higher sclerostin levels are causally related to lower BMD and greater fracture risk. Hence, strategies for reducing circulating sclerostin, for example by targeting glycosylation enzymes as suggested by our GWAS results, may prove valuable in treating osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jbmr.3803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899787PMC
October 2019

Genetic Polymorphism of miR-196a-2 is Associated with Bone Mineral Density (BMD).

Int J Mol Sci 2017 Nov 25;18(12). Epub 2017 Nov 25.

Department of Epidemiology, Erasmus University Medical Center, 's-Gravendijkwal 230, 3015 CE Rotterdam, the Netherlands.

MicroRNAs (miRNAs) are small non-coding RNA molecules that post-transcriptionally regulate the translation of messenger RNAs. Given the crucial role of miRNAs in gene expression, genetic variants within miRNA-related sequences may affect miRNA function and contribute to disease risk. Osteoporosis is characterized by reduced bone mass, and bone mineral density (BMD) is a major diagnostic proxy to assess osteoporosis risk. Here, we aimed to identify miRNAs that are involved in BMD using data from recent genome-wide association studies (GWAS) on femoral neck, lumbar spine and forearm BMD. Of 242 miRNA-variants available in the GWAS data, we found rs11614913:C > T in the precursor to be significantly associated with femoral neck-BMD (-value = 9.9 × 10, β = -0.038) and lumbar spine-BMD (-value = 3.2 × 10, β = -0.061). Furthermore, our sensitivity analyses using the Rotterdam study data showed a sex-specific association of rs11614913 with BMD only in women. Subsequently, we highlighted a number of target genes, expressed in bone and associated with BMD, that may mediate the miRNA function in BMD. Collectively, our results suggest that may contribute to variations in BMD level. Further biological investigations will give more insights into the mechanisms by which control expression of BMD-related genes.
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http://dx.doi.org/10.3390/ijms18122529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751132PMC
November 2017

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density.

Ann Rheum Dis 2018 03 23;77(3):378-385. Epub 2017 Nov 23.

Rheumatology and Bone Disease Unit, Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Objectives: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis.

Methods: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies.

Results: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes (tubulin tyrosine ligase) and (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures.

Conclusion: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism.
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http://dx.doi.org/10.1136/annrheumdis-2017-212469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912156PMC
March 2018

Bivariate genome-wide association meta-analysis of pediatric musculoskeletal traits reveals pleiotropic effects at the SREBF1/TOM1L2 locus.

Nat Commun 2017 07 25;8(1):121. Epub 2017 Jul 25.

Department of Internal Medicine, Erasmus MC University, Rotterdam, 3015GE, The Netherlands.

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% (95% CI: 34-52%) for TBLH-BMD, and 39% (95% CI: 30-48%) for TB-LM, with a shared genetic component of 43% (95% CI: 29-56%). We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5. Variants in the TOM1L2/SREBF1 locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that SREBF1 is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass.Bone mineral density and lean skeletal mass are heritable traits. Here, Medina-Gomez and colleagues perform bivariate GWAS analyses of total body lean mass and bone mass density in children, and show genetic loci with pleiotropic effects on both traits.
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http://dx.doi.org/10.1038/s41467-017-00108-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527106PMC
July 2017

Distinct DNA methylation profiles in bone and blood of osteoporotic and healthy postmenopausal women.

Epigenetics 2017 08 26;12(8):674-687. Epub 2017 Jun 26.

b Lovisenberg Diakonale Hospital, Unger-Vetlesen Institute , Oslo , Norway.

DNA methylation affects expression of associated genes and may contribute to the missing genetic effects from genome-wide association studies of osteoporosis. To improve insight into the mechanisms of postmenopausal osteoporosis, we combined transcript profiling with DNA methylation analyses in bone. RNA and DNA were isolated from 84 bone biopsies of postmenopausal donors varying markedly in bone mineral density (BMD). In all, 2529 CpGs in the top 100 genes most significantly associated with BMD were analyzed. The methylation levels at 63 CpGs differed significantly between healthy and osteoporotic women at 10% false discovery rate (FDR). Five of these CpGs at 5% FDR could explain 14% of BMD variation. To test whether blood DNA methylation reflect the situation in bone (as shown for other tissues), an independent cohort was selected and BMD association was demonstrated in blood for 13 of the 63 CpGs. Four transcripts representing inhibitors of bone metabolism-MEPE, SOST, WIF1, and DKK1-showed correlation to a high number of methylated CpGs, at 5% FDR. Our results link DNA methylation to the genetic influence modifying the skeleton, and the data suggest a complex interaction between CpG methylation and gene regulation. This is the first study in the hitherto largest number of postmenopausal women to demonstrate a strong association among bone CpG methylation, transcript levels, and BMD/fracture. This new insight may have implications for evaluation of osteoporosis stage and susceptibility.
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http://dx.doi.org/10.1080/15592294.2017.1345832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687328PMC
August 2017

Omics analysis of human bone to identify genes and molecular networks regulating skeletal remodeling in health and disease.

Bone 2017 Aug 24;101:88-95. Epub 2017 Apr 24.

Lovisenberg Diakonale Hospital, Unger-Vetlesen Institute, Oslo, Norway; University of Oslo, Institute of Basic Medical Sciences, Oslo, Norway.

The skeleton is a metabolically active organ throughout life where specific bone cell activity and paracrine/endocrine factors regulate its morphogenesis and remodeling. In recent years, an increasing number of reports have used multi-omics technologies to characterize subsets of bone biological molecular networks. The skeleton is affected by primary and secondary disease, lifestyle and many drugs. Therefore, to obtain relevant and reliable data from well characterized patient and control cohorts are vital. Here we provide a brief overview of omics studies performed on human bone, of which our own studies performed on trans-iliacal bone biopsies from postmenopausal women with osteoporosis (OP) and healthy controls are among the first and largest. Most other studies have been performed on smaller groups of patients, undergoing hip replacement for osteoarthritis (OA) or fracture, and without healthy controls. The major findings emerging from the combined studies are: 1. Unstressed and stressed bone show profoundly different gene expression reflecting differences in bone turnover and remodeling and 2. Omics analyses comparing healthy/OP and control/OA cohorts reveal characteristic changes in transcriptomics, epigenomics (DNA methylation), proteomics and metabolomics. These studies, together with genome-wide association studies, in vitro observations and transgenic animal models have identified a number of genes and gene products that act via Wnt and other signaling systems and are highly associated to bone density and fracture. Future challenge is to understand the functional interactions between bone-related molecular networks and their significance in OP and OA pathogenesis, and also how the genomic architecture is affected in health and disease.
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http://dx.doi.org/10.1016/j.bone.2017.04.012DOI Listing
August 2017

Novel Genetic Variants Associated With Increased Vertebral Volumetric BMD, Reduced Vertebral Fracture Risk, and Increased Expression of SLC1A3 and EPHB2.

J Bone Miner Res 2016 12 6;31(12):2085-2097. Epub 2016 Sep 6.

Institute for Aging Research, Hebrew SeniorLife, Harvard Medical School, Boston, MA, USA.

Genome-wide association studies (GWASs) have revealed numerous loci for areal bone mineral density (aBMD). We completed the first GWAS meta-analysis (n = 15,275) of lumbar spine volumetric BMD (vBMD) measured by quantitative computed tomography (QCT), allowing for examination of the trabecular bone compartment. SNPs that were significantly associated with vBMD were also examined in two GWAS meta-analyses to determine associations with morphometric vertebral fracture (n = 21,701) and clinical vertebral fracture (n = 5893). Expression quantitative trait locus (eQTL) analyses of iliac crest biopsies were performed in 84 postmenopausal women, and murine osteoblast expression of genes implicated by eQTL or by proximity to vBMD-associated SNPs was examined. We identified significant vBMD associations with five loci, including: 1p36.12, containing WNT4 and ZBTB40; 8q24, containing TNFRSF11B; and 13q14, containing AKAP11 and TNFSF11. Two loci (5p13 and 1p36.12) also contained associations with radiographic and clinical vertebral fracture, respectively. In 5p13, rs2468531 (minor allele frequency [MAF] = 3%) was associated with higher vBMD (β = 0.22, p = 1.9 × 10 ) and decreased risk of radiographic vertebral fracture (odds ratio [OR] = 0.75; false discovery rate [FDR] p = 0.01). In 1p36.12, rs12742784 (MAF = 21%) was associated with higher vBMD (β = 0.09, p = 1.2 × 10 ) and decreased risk of clinical vertebral fracture (OR = 0.82; FDR p = 7.4 × 10 ). Both SNPs are noncoding and were associated with increased mRNA expression levels in human bone biopsies: rs2468531 with SLC1A3 (β = 0.28, FDR p = 0.01, involved in glutamate signaling and osteogenic response to mechanical loading) and rs12742784 with EPHB2 (β = 0.12, FDR p = 1.7 × 10 , functions in bone-related ephrin signaling). Both genes are expressed in murine osteoblasts. This is the first study to link SLC1A3 and EPHB2 to clinically relevant vertebral osteoporosis phenotypes. These results may help elucidate vertebral bone biology and novel approaches to reducing vertebral fracture incidence. © 2016 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.2913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477772PMC
December 2016

The Influence of DNA Methylation on Bone Cells.

Curr Genomics 2015 Dec;16(6):384-92

Lovisenberg Diakonale Hospital, Oslo, Norway;; University of Oslo, Institute of Basic Medical Sciences, Oslo, Norway;

DNA methylation in eukaryotes invokes heritable alterations of the of the cytosine base in DNA without changing the underlying genomic DNA sequence. DNA methylation may be modified by environmental exposures as well as gene polymorphisms and may be a mechanistic link between environmental risk factors and the development of disease. In this review, we consider the role of DNA methylation in bone cells (osteoclasts/osteoblasts/osteocytes) and their progenitors with special focus on in vitro and ex vivo analyses. The number of studies on DNA methylation in bone cells is still somewhat limited, nevertheless it is getting increasingly clear that this type of the epigenetic changes is a critical regulator of gene expression. DNA methylation is necessary for proper development and function of bone cells and is accompanied by disease characteristic functional alterations as presently reviewed including postmenopausal osteoporosis and mechanical strain.
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http://dx.doi.org/10.2174/1389202916666150817202913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765525PMC
December 2015

Genetic Sharing with Cardiovascular Disease Risk Factors and Diabetes Reveals Novel Bone Mineral Density Loci.

PLoS One 2015 22;10(12):e0144531. Epub 2015 Dec 22.

NORMENT, KG Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.

Bone Mineral Density (BMD) is a highly heritable trait, but genome-wide association studies have identified few genetic risk factors. Epidemiological studies suggest associations between BMD and several traits and diseases, but the nature of the suggestive comorbidity is still unknown. We used a novel genetic pleiotropy-informed conditional False Discovery Rate (FDR) method to identify single nucleotide polymorphisms (SNPs) associated with BMD by leveraging cardiovascular disease (CVD) associated disorders and metabolic traits. By conditioning on SNPs associated with the CVD-related phenotypes, type 1 diabetes, type 2 diabetes, systolic blood pressure, diastolic blood pressure, high density lipoprotein, low density lipoprotein, triglycerides and waist hip ratio, we identified 65 novel independent BMD loci (26 with femoral neck BMD and 47 with lumbar spine BMD) at conditional FDR < 0.01. Many of the loci were confirmed in genetic expression studies. Genes validated at the mRNA levels were characteristic for the osteoblast/osteocyte lineage, Wnt signaling pathway and bone metabolism. The results provide new insight into genetic mechanisms of variability in BMD, and a better understanding of the genetic underpinnings of clinical comorbidity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144531PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687843PMC
June 2016

Identifying Novel Gene Variants in Coronary Artery Disease and Shared Genes With Several Cardiovascular Risk Factors.

Circ Res 2016 Jan 20;118(1):83-94. Epub 2015 Oct 20.

From the Department of Clinical Molecular Biology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway (M.L., B.K.A.); Oslo Centre for Biostatistics and Epidemiology, Department of Biostatistics, University of Oslo, and Research Support Services, Oslo University Hospital, Oslo, Norway (M.L., A.F.); NORMENT - K.G. Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University of Oslo, Oslo, Norway (V.Z., A.W., F.B., Y.W., S.D., O.A.A.); Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway (V.Z., A.W., F.B., S.D., O.A.A.); Oslo Centre for Biostatistics and Epidemiology, Department of Biostatistics, University of Oslo, Oslo, Norway (B.K.A.); Deutsches Herzzentrum München, Technische Universität München, Munich, Germany (L.Z., H.S.); Deutsches Zentrum für Herz-Kreislauf-Forschung, partner site Munich Heart Alliance, Munich, Germany (L.Z., H.S.); Multimodal Imaging Laboratory, University of California at San Diego, La Jolla (Y.W., L.K.M., A.J.S., R.S.D., A.M.D., O.A.A.); Department of Neurosciences, University of California, San Diego, La Jolla, (Y.W., A.M.D.); Department of Radiology, University of California, San Diego, La Jolla (L.K.M., R.S.D., A.M.D.); Department of Psychiatry, University of California, San Diego, La Jolla (W.K.T., A.M.D.); Cognitive Sciences Graduate Program, University of California, San Diego, La Jolla, (A.J.S.); Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway (S.R.); Department of Medical Biochemistry, Lovisenberg Diakonale Hospital, Oslo, Norway (S.R., K.M.G.); Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway (S.R., K.M.G.); Family and Preventive Medicine, Division of Epidemiology, University of California, San Diego, La Jolla (E.B.-C.); Department of Epidemiology, Erasmus University Medical Center, Rotterdam, The Netherlands (S.L., A.D.); Department of Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom (C.P.N.,

Rationale: Coronary artery disease (CAD) is a critical determinant of morbidity and mortality. Previous studies have identified several cardiovascular disease risk factors, which may partly arise from a shared genetic basis with CAD, and thus be useful for discovery of CAD genes.

Objective: We aimed to improve discovery of CAD genes and inform the pathogenic relationship between CAD and several cardiovascular disease risk factors using a shared polygenic signal-informed statistical framework.

Methods And Results: Using genome-wide association studies summary statistics and shared polygenic pleiotropy-informed conditional and conjunctional false discovery rate methodology, we systematically investigated genetic overlap between CAD and 8 traits related to cardiovascular disease risk factors: low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, type 2 diabetes mellitus, C-reactive protein, body mass index, systolic blood pressure, and type 1 diabetes mellitus. We found significant enrichment of single-nucleotide polymorphisms associated with CAD as a function of their association with low-density lipoprotein, high-density lipoprotein, triglycerides, type 2 diabetes mellitus, C-reactive protein, body mass index, systolic blood pressure, and type 1 diabetes mellitus. Applying the conditional false discovery rate method to the enriched phenotypes, we identified 67 novel loci associated with CAD (overall conditional false discovery rate <0.01). Furthermore, we identified 53 loci with significant effects in both CAD and at least 1 of low-density lipoprotein, high-density lipoprotein, triglycerides, type 2 diabetes mellitus, C-reactive protein, systolic blood pressure, and type 1 diabetes mellitus.

Conclusions: The observed polygenic overlap between CAD and cardiometabolic risk factors indicates a pathogenic relation that warrants further investigation. The new gene loci identified implicate novel genetic mechanisms related to CAD.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.306629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609485PMC
January 2016

The α2β1 binding domain of chondroadherin inhibits breast cancer-induced bone metastases and impairs primary tumour growth: a preclinical study.

Cancer Lett 2015 Mar 18;358(1):67-75. Epub 2014 Dec 18.

Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy.

cyclicCHAD is a peptide representing the α2β1 integrin binding sequence of the matrix protein chondroadherin (CHAD), which in our hands proved effective at counteracting bone loss in ovariectomised mice by inhibiting osteoclastogenesis. Given that bone metastases are characterised by exacerbated osteoclast activity as well, we tested this therapy in mice intracardiacally injected with the osteotropic human breast cancer cell line MDA-MB-231. Treatment with cyclicCHAD significantly decreased cachexia and incidence of bone metastases, and induced a trend of reduction of visceral metastasis volume, while in orthotopically injected mice cyclicCHAD reduced tumour volume. In vitro studies showed its ability to impair tumour cell motility and invasion, suggesting a direct effect not only on osteoclasts but also on the tumour cell phenotype. Interestingly, when administered together with a suboptimal, poorly effective, dose of doxorubicin (DXR), cyclicCHAD improved survival and reduced visceral metastases volume to a level similar to that of the optimal dose of DXR alone. Taken together, these preclinical data suggest that cyclicCHAD is a new inhibitor of bone metastases, with an appreciable direct effect also on tumour growth and a synergistic activity in combination with low dose chemotherapy, underscoring an important translational impact.
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http://dx.doi.org/10.1016/j.canlet.2014.12.032DOI Listing
March 2015

Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women.

J Bone Miner Res 2015 Feb;30(2):249-56

Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; Lovisenberg Diakonale Hospital, Oslo, Norway; Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T-score > -1) and established OP (BMD T-score < -2.5, with at least one low-energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (n = 4) compared to age and body mass index (BMI) balanced controls (n = 4) (80.5% versus 63.2%, p = 0.0001) with replication in independent cohorts (n = 27 and n = 36, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age-adjusted and BMI-adjusted total hip BMD (r = 0.47 and r = 0.43, respectively; both p < 0.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population-based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (p = 0.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation.
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http://dx.doi.org/10.1002/jbmr.2342DOI Listing
February 2015

The C-terminal domain of chondroadherin: a new regulator of osteoclast motility counteracting bone loss.

J Bone Miner Res 2014 Aug;29(8):1833-46

Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy.

Chondroadherin (CHAD) is a leucine-rich protein promoting cell attachment through binding to integrin α2 β1 and syndecans. We observed that CHAD mRNA and protein were lower in bone biopsies of 50-year-old to 65-year-old osteoporotic women and in bone samples of ovariectomized mice versus gender/age-matched controls, suggesting a role in bone metabolism. By the means of an internal cyclic peptide (cyclicCHAD), we observed that its integrin binding sequence impaired preosteoclast migration through a nitric oxide synthase 2-dependent mechanism, decreasing osteoclastogenesis and bone resorption in a concentration-dependent fashion, whereas it had no effect on osteoblasts. Consistently, cyclicCHAD reduced transcription of two nitric oxide downstream genes, migfilin and vasp, involved in cell motility. Furthermore, the nitric oxide donor, S-nitroso-N-acetyl-D,L-penicillamine, stimulated preosteoclast migration and prevented the inhibitory effect of cyclicCHAD. Conversely, the nitric oxide synthase 2 (NOS2) inhibitor, N5-(1-iminoethyl)-l-ornithine, decreased both preosteoclast migration and differentiation, confirming a role of the nitric oxide pathway in the mechanism of action triggered by cyclicCHAD. In vivo, administration of cyclicCHAD was well tolerated and increased bone volume in healthy mice, with no adverse effect. In ovariectomized mice cyclicCHAD improved bone mass by both a preventive and a curative treatment protocol, with an effect in line with that of the bisphosphonate alendronate, that was mimicked by the NOS2 inhibitor [L-N6-(1-Iminoethyl)-lysine.2 dihydrochloride]. In both mouse models, cyclicCHAD reduced osteoclast and bone resorption without affecting osteoblast parameters and bone formation. In conclusion, CHAD is a novel regulator of bone metabolism that, through its integrin binding domain, inhibits preosteoclast motility and bone resorption, with a potential translational impact for the treatment of osteoporosis.
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http://dx.doi.org/10.1002/jbmr.2206DOI Listing
August 2014

Global miRNA expression analysis of serous and clear cell ovarian carcinomas identifies differentially expressed miRNAs including miR-200c-3p as a prognostic marker.

BMC Cancer 2014 Feb 11;14:80. Epub 2014 Feb 11.

Department of Gynecological Oncology, Oslo University Hospital (OUH), The Norwegian Radium Hospital, Postbox 4953 Nydalen 0424, Oslo, Norway.

Background: Improved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions.

Methods: Differences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously.

Results: Differentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC.

Conclusions: Several miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped.
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http://dx.doi.org/10.1186/1471-2407-14-80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928323PMC
February 2014

Transcriptional profiling of mRNAs and microRNAs in human bone marrow precursor B cells identifies subset- and age-specific variations.

PLoS One 2013 30;8(7):e70721. Epub 2013 Jul 30.

Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.

Background: Molecular mechanisms explaining age-related changes in the bone marrow with reduced precursor B cell output are poorly understood.

Methods: We studied the transcriptome of five precursor B cell subsets in individual bone marrow samples from 4 healthy children and 4 adults employing GeneChip® Human Exon 1.0 ST Arrays (Affymetrix®) and TaqMan® Array MicroRNA Cards (Life Technologies™).

Results: A total of 1796 mRNAs (11%) were at least once differentially expressed between the various precursor B cell subsets in either age group (FDR 0.1%, p≤1.13×10(-4)) with more marked cell stage specific differences than those related to age. In contrast, microRNA profiles of the various precursor B cell subsets showed less hierarchical clustering as compared to the corresponding mRNA profiles. However, 17 of the 667 microRNA assays (2.5%) were at least once differentially expressed between the subsets (FDR 10%, p≤0.004). From target analysis (Ingenuity® Systems), functional assignment between postulated interacting mRNAs and microRNAs showed especially association to cellular growth, proliferation and cell cycle regulation. One functional network connected up-regulation of the differentiation inhibitor ID2 mRNA to down-regulation of the hematopoiesis- or cell cycle regulating miR-125b-5p, miR-181a-5p, miR-196a-5p, miR-24-3p and miR-320d in adult PreBII large cells. Noteworthy was also the stage-dependent expression of the growth promoting miR-17-92 cluster, showing a partly inverse trend with age, reaching statistical significance at the PreBII small stage (up 3.1-12.9 fold in children, p = 0.0084-0.0270).

Conclusions: The global mRNA profile is characteristic for each precursor B cell developmental stage and largely similar in children and adults. The microRNA profile is much cell stage specific and not changing much with age. Importantly, however, specific age-dependent differences involving key networks like differentiation and cellular growth may indicate biological divergence and possibly also altered production potential with age.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070721PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728296PMC
April 2014

Increased ID2 levels in adult precursor B cells as compared with children is associated with impaired Ig locus contraction and decreased bone marrow output.

J Immunol 2013 Aug 3;191(3):1210-9. Epub 2013 Jul 3.

Department of Medical Biochemistry, Oslo University Hospital, 0407 Oslo, Norway.

Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation-stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA-PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdT was 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2.
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http://dx.doi.org/10.4049/jimmunol.1203462DOI Listing
August 2013

Identification of transcriptional macromolecular associations in human bone using browser based in silico analysis in a giant correlation matrix.

Bone 2013 Mar 27;53(1):69-78. Epub 2012 Nov 27.

Department of Medical Biochemistry, Oslo University Hospital, Ullevaal, Norway.

Intracellular signaling is critically dependent on gene regulatory networks comprising physical molecular interactions. Presently, there is a lack of comprehensive databases for most human tissue types to verify such macromolecular interactions. We present a user friendly browser which helps to identify functional macromolecular interactions in human bone as significant correlations at the transcriptional level. The molecular skeletal phenotype has been characterized by transcriptome analysis of iliac crest bone biopsies from 84 postmenopausal women through quantifications of ~23,000 mRNA species. When the signal levels were inter-correlated, an array containing >260 million correlations was generated, thus recognizing the human bone interactome at the RNA level. The matrix correlation and p values were made easily accessible by a freely available online browser. We show that significant correlations within the giant matrix are reproduced in a replica set of 13 male vertebral biopsies. The identified correlations differ somewhat from transcriptional interactions identified in cell culture experiments and transgenic mice, thus demonstrating that care should be taken in extrapolating such results to the in vivo situation in human bone. The current giant matrix and web browser are a valuable tool for easy access to the human bone transcriptome and molecular interactions represented as significant correlations at the RNA-level. The browser and matrix should be a valuable hypothesis generating tool for identification of regulatory mechanisms and serve as a library of transcript relationships in human bone, a relatively inaccessible tissue.
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http://dx.doi.org/10.1016/j.bone.2012.11.015DOI Listing
March 2013

ZNF385B and VEGFA are strongly differentially expressed in serous ovarian carcinomas and correlate with survival.

PLoS One 2012 28;7(9):e46317. Epub 2012 Sep 28.

Department of Gynaecological Oncology, Oslo University Hospital, Oslo, Norway.

Background: The oncogenesis of ovarian cancer is poorly understood. The aim of this study was to identify mRNAs differentially expressed between moderately and poorly differentiated (MD/PD) serous ovarian carcinomas (SC), serous ovarian borderline tumours (SBOT) and superficial scrapings from normal ovaries (SNO), and to correlate these mRNAs with clinical parameters including survival.

Methods: Differences in mRNA expression between MD/PD SC, SBOT and SNO were analyzed by global gene expression profiling (n = 23), validated by RT-qPCR (n = 41) and correlated with clinical parameters.

Results: Thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected from the global gene expression analyses, and 21 were verified (p<0.01) by RT-qPCR. Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. ZNF385B was downregulated (FC = -130.5, p = 1.2×10(-7)) and correlated with overall survival (p = 0.03). VEGFA was upregulated (FC = 6.1, p = 6.0×10(-6)) and correlated with progression-free survival (p = 0.037). Increased levels of TPX2 and FOXM1 mRNAs (FC = 28.5, p = 2.7×10(-10) and FC = 46.2, p = 5.6×10(-4), respectively) correlated with normalization of CA125 (p = 0.03 and p = 0.044, respectively). Furthermore, we present a molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and directly interacting with TP53.

Conclusions: We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including ZNF385B and VEGFA correlating with survival, and FOXM1 and TPX2 with normalization of CA125. We also present a molecular pathway for MD/PD SC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460818PMC
February 2013

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus.

PLoS Genet 2012 Jul 5;8(7):e1002718. Epub 2012 Jul 5.

Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands.

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.
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http://dx.doi.org/10.1371/journal.pgen.1002718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390371PMC
July 2012

Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture.

Authors:
Karol Estrada Unnur Styrkarsdottir Evangelos Evangelou Yi-Hsiang Hsu Emma L Duncan Evangelia E Ntzani Ling Oei Omar M E Albagha Najaf Amin John P Kemp Daniel L Koller Guo Li Ching-Ti Liu Ryan L Minster Alireza Moayyeri Liesbeth Vandenput Dana Willner Su-Mei Xiao Laura M Yerges-Armstrong Hou-Feng Zheng Nerea Alonso Joel Eriksson Candace M Kammerer Stephen K Kaptoge Paul J Leo Gudmar Thorleifsson Scott G Wilson James F Wilson Ville Aalto Markku Alen Aaron K Aragaki Thor Aspelund Jacqueline R Center Zoe Dailiana David J Duggan Melissa Garcia Natàlia Garcia-Giralt Sylvie Giroux Göran Hallmans Lynne J Hocking Lise Bjerre Husted Karen A Jameson Rita Khusainova Ghi Su Kim Charles Kooperberg Theodora Koromila Marcin Kruk Marika Laaksonen Andrea Z Lacroix Seung Hun Lee Ping C Leung Joshua R Lewis Laura Masi Simona Mencej-Bedrac Tuan V Nguyen Xavier Nogues Millan S Patel Janez Prezelj Lynda M Rose Serena Scollen Kristin Siggeirsdottir Albert V Smith Olle Svensson Stella Trompet Olivia Trummer Natasja M van Schoor Jean Woo Kun Zhu Susana Balcells Maria Luisa Brandi Brendan M Buckley Sulin Cheng Claus Christiansen Cyrus Cooper George Dedoussis Ian Ford Morten Frost David Goltzman Jesús González-Macías Mika Kähönen Magnus Karlsson Elza Khusnutdinova Jung-Min Koh Panagoula Kollia Bente Lomholt Langdahl William D Leslie Paul Lips Östen Ljunggren Roman S Lorenc Janja Marc Dan Mellström Barbara Obermayer-Pietsch José M Olmos Ulrika Pettersson-Kymmer David M Reid José A Riancho Paul M Ridker François Rousseau P Eline Slagboom Nelson L S Tang Roser Urreizti Wim Van Hul Jorma Viikari María T Zarrabeitia Yurii S Aulchenko Martha Castano-Betancourt Elin Grundberg Lizbeth Herrera Thorvaldur Ingvarsson Hrefna Johannsdottir Tony Kwan Rui Li Robert Luben Carolina Medina-Gómez Stefan Th Palsson Sjur Reppe Jerome I Rotter Gunnar Sigurdsson Joyce B J van Meurs Dominique Verlaan Frances M K Williams Andrew R Wood Yanhua Zhou Kaare M Gautvik Tomi Pastinen Soumya Raychaudhuri Jane A Cauley Daniel I Chasman Graeme R Clark Steven R Cummings Patrick Danoy Elaine M Dennison Richard Eastell John A Eisman Vilmundur Gudnason Albert Hofman Rebecca D Jackson Graeme Jones J Wouter Jukema Kay-Tee Khaw Terho Lehtimäki Yongmei Liu Mattias Lorentzon Eugene McCloskey Braxton D Mitchell Kannabiran Nandakumar Geoffrey C Nicholson Ben A Oostra Munro Peacock Huibert A P Pols Richard L Prince Olli Raitakari Ian R Reid John Robbins Philip N Sambrook Pak Chung Sham Alan R Shuldiner Frances A Tylavsky Cornelia M van Duijn Nick J Wareham L Adrienne Cupples Michael J Econs David M Evans Tamara B Harris Annie Wai Chee Kung Bruce M Psaty Jonathan Reeve Timothy D Spector Elizabeth A Streeten M Carola Zillikens Unnur Thorsteinsdottir Claes Ohlsson David Karasik J Brent Richards Matthew A Brown Kari Stefansson André G Uitterlinden Stuart H Ralston John P A Ioannidis Douglas P Kiel Fernando Rivadeneira

Nat Genet 2012 Apr 15;44(5):491-501. Epub 2012 Apr 15.

Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands.

Bone mineral density (BMD) is the most widely used predictor of fracture risk. We performed the largest meta-analysis to date on lumbar spine and femoral neck BMD, including 17 genome-wide association studies and 32,961 individuals of European and east Asian ancestry. We tested the top BMD-associated markers for replication in 50,933 independent subjects and for association with risk of low-trauma fracture in 31,016 individuals with a history of fracture (cases) and 102,444 controls. We identified 56 loci (32 new) associated with BMD at genome-wide significance (P < 5 × 10(-8)). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal stem cell differentiation, endochondral ossification and Wnt signaling pathways. However, we also discovered loci that were localized to genes not known to have a role in bone biology. Fourteen BMD-associated loci were also associated with fracture risk (P < 5 × 10(-4), Bonferroni corrected), of which six reached P < 5 × 10(-8), including at 18p11.21 (FAM210A), 7q21.3 (SLC25A13), 11q13.2 (LRP5), 4q22.1 (MEPE), 2p16.2 (SPTBN1) and 10q21.1 (DKK1). These findings shed light on the genetic architecture and pathophysiological mechanisms underlying BMD variation and fracture susceptibility.
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http://dx.doi.org/10.1038/ng.2249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338864PMC
April 2012

Cerebrospinal fluid hypocretin 1 deficiency, overweight, and metabolic dysregulation in patients with narcolepsy.

J Clin Sleep Med 2011 Dec;7(6):653-8

Department of Clinical Neurophysiology, Oslo University Hospital, Ullevål, Oslo, Norway.

Study Objectives: The possible relationship between cerebrospinal fluid (CSF) hypocretin and leptin levels, overweight, and association to risk factors for diabetes 2 in narcolepsy with cataplexy were compared to patients with idiopathic hypersomnia and controls.

Patients: 26 patients with narcolepsy, cataplexy, and hypocretin deficiency; 23 patients with narcolepsy, cataplexy, and normal hypocretin values; 11 patients with idiopathic hypersomnia; and 43 controls.

Measurements And Results: Body mass index (BMI), serum leptin, and HbA1C were measured in patients and controls; and CSF hypocretin 1 and leptin measured in all patients. Female and male patients with narcolepsy and hypocretin deficiency had the highest mean BMI (27.8 and 26.2, respectively), not statistically different from patients with narcolepsy and normal hypocretin or controls, but statistically higher than the patients with idiopathic hypersomnia (p < 0.001 and 0.011, respectively). The number of obese patients (BMI > 30) was increased in both narcolepsy groups. Serum and CSF leptin levels correlated positively to BMI in patients and controls, but not to CSF hypocretin concentrations. HbA1C was within normal levels and similar in all groups.

Conclusions: The study confirms a moderate tendency to obesity (BMI > 30) and overweight in patients with narcolepsy and cataplexy. Obesity was not correlated to hypocretin deficiency or reduced serum or CSF leptin concentrations. We suggest that overweight and possible metabolic changes previously reported in narcolepsy, may be caused by other mechanisms.
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http://dx.doi.org/10.5664/jcsm.1474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227712PMC
December 2011

Molecular disease map of bone characterizing the postmenopausal osteoporosis phenotype.

J Bone Miner Res 2011 Aug;26(8):1793-801

Section of Endocrinology, Department of Medicine, Rikshospitalet University Hospital, Oslo, Norway.

Genome-wide gene expressions in bone biopsies from patients with postmenopausal osteoporosis and healthy controls were profiled, to identify osteoporosis candidate genes. All osteoporotic patients (n = 27) in an unbiased cohort of Norwegian women presented with bone mineral density (BMD) T-scores of less than -2.5 SD and one or more confirmed low-energy fracture(s). A validation group (n = 18) had clinical and laboratory parameters intermediate to the control (n = 39) and osteoporosis groups. RNA from iliac crest bone biopsies were analyzed by Affymetrix microarrays and real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Differentially expressed genes in osteoporosis versus control groups were identified using the Bayesian ANOVA for microarrays (BAMarray) method, whereas the R-package Limma (Linear Models for Microarray Data) was used to determine whether these transcripts were explained by disease, age, body mass index (BMI), or combinations thereof. Laboratory tests showed normal ranges for the cohort. A total of 609 transcripts were differentially expressed in osteoporotic patients relative to controls; 256 transcripts were confirmed for disease when controlling for age or BMI. Most of the osteoporosis susceptibility genes (80%) also were confirmed to be regulated in the same direction in the validation group. Furthermore, 217 of 256 transcripts were correlated with BMD (adjusted for age and BMI) at various skeletal sites (|r| > 0.2, p < .05). Among the most distinctly expressed genes were Wnt antagonists DKK1 and SOST, the transcription factor SOX4, and the bone matrix proteins MMP13 and MEPE, all reduced in osteoporosis versus control groups. Our results identify potential osteoporosis susceptibility candidate genes adjusted for confounding factors (ie, age and BMI) with or without a significant correlation with BMD.
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http://dx.doi.org/10.1002/jbmr.396DOI Listing
August 2011

POLD2 and KSP37 (FGFBP2) correlate strongly with histology, stage and outcome in ovarian carcinomas.

PLoS One 2010 Nov 4;5(11):e13837. Epub 2010 Nov 4.

Department of Gynaecology, Oslo University Hospital, Oslo, Norway.

Background: Epithelial ovarian cancer (EOC) constitutes more than 90% of ovarian cancers and is associated with high mortality. EOC comprises a heterogeneous group of tumours, and the causes and molecular pathology are essentially unknown. Improved insight into the molecular characteristics of the different subgroups of EOC is urgently needed, and should eventually lead to earlier diagnosis as well as more individualized and effective treatments. Previously, we reported a limited number of mRNAs strongly upregulated in human osteosarcomas and other malignancies, and six were selected to be tested for a possible association with three subgroups of ovarian carcinomas and clinical parameters.

Methodology/principal Findings: The six selected mRNAs were quantified by RT-qPCR in biopsies from eleven poorly differentiated serous carcinomas (PDSC, stage III-IV), twelve moderately differentiated serous carcinomas (MDSC, stage III-IV) and eight clear cell carcinomas (CCC, stage I-IV) of the ovary. Superficial scrapings from six normal ovaries (SNO), as well as biopsies from three normal ovaries (BNO) and three benign ovarian cysts (BBOC) were analyzed for comparison. The gene expression level was related to the histological and clinical parameters of human ovarian carcinoma samples. One of the mRNAs, DNA polymerase delta 2 small subunit (POLD2), was increased in average 2.5- to almost 20-fold in MDSC and PDSC, respectively, paralleling the degree of dedifferentiation and concordant with a poor prognosis. Except for POLD2, the serous carcinomas showed a similar transcription profile, being clearly different from CCC. Another mRNA, Killer-specific secretory protein of 37 kDa (KSP37) showed six- to eight-fold higher levels in CCC stage I compared with the more advanced staged carcinomas, and correlated positively with an improved clinical outcome.

Conclusions/significance: We have identified two biomarkers which are markedly upregulated in two subgroups of ovarian carcinomas and are also associated with stage and outcome. The results suggest that POLD2 and KSP37 might be potential prognostic biomarkers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013837PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2973954PMC
November 2010

Periostin is a collagen associated bone matrix protein regulated by parathyroid hormone.

Matrix Biol 2010 Sep 21;29(7):594-601. Epub 2010 Jul 21.

Department of Biochemistry, Institute of Basic Medical Sciences, PO Box 1112 Blindern, University of Oslo, N-0317 Oslo, Norway.

Periostin is a 90 kDa secreted protein, originally identified in murine osteoblast-like cells, with a distribution restricted to collagen-rich tissues and certain tumors. In this paper, we first analyzed the expression of periostin mRNA and protein in human fetal osteoblasts (hFOB) and human osteosarcoma (hOS) cell lines by RT real-time PCR and Western blot, respectively. The hFOB 1.19 and three hOS (MHM, KPDXM and Eggen) showed highly variable periostin mRNA levels and protein. Second, we showed that the expression of periostin mRNA was inversely related to the cells' abilities to differentiate and mineralize. Then, we investigated the regulation of periostin mRNA in hFOB after siRNA treatment and in mouse primary osteoblasts (mOB) treated with PTH. Knock-down of periostin mRNA, down-regulated PTHrP, but did not affect the expression of other important markers of differentiation such as RUNX2. In addition, periostin mRNA was transiently up-regulated in osteoblasts by PTH. Finally, the localization of periostin and its partially co-localization with collagen 1a1 mRNA and protein was studied in mouse embryos and postnatal pups using in situ hybridization and immunohistochemistry, respectively. In conclusion, the present study provides novel observations related to the expression, distribution and regulation of periostin in bone cells and extracellular matrix.
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http://dx.doi.org/10.1016/j.matbio.2010.07.001DOI Listing
September 2010

Skeletal site-related variation in human trabecular bone transcriptome and signaling.

PLoS One 2010 May 18;5(5):e10692. Epub 2010 May 18.

Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle Upon Tyne, United Kingdom.

Background: The skeletal site-specific influence of multiple genes on bone morphology is recognised, but the question as to how these influences may be exerted at the molecular and cellular level has not been explored.

Methodology: To address this question, we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina.

Principal Findings: In the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. This analysis has also led to the identification of expression of a number of transcripts, previously known and novel, which to our knowledge have never earlier been associated with bone growth and remodelling.

Conclusions And Significance: This study provides molecular evidence explaining anatomical and micro-architectural site-related changes in bone cell function, which is predominantly attributable to alteration in cell transcriptional activity. A number of novel signaling molecules in critical pathways, which have been hitherto not known to be expressed in bone cells of mature vertebrates, were identified.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010692PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872667PMC
May 2010

Zic1 transcription factor in bone: neural developmental protein regulates mechanotransduction in osteocytes.

FASEB J 2010 Aug 30;24(8):2893-903. Epub 2010 Mar 30.

Musculoskeletal Research Group, ICM, The Medical School, Newcastle University, Newcastle upon Tyne, UK.

A transcriptome analysis compared gene expression in human bone biopsy samples taken from lumbar spine and iliac crest, sites that experience high and low levels of mechanical stress, respectively. The analysis revealed that the zinc finger protein of cerebellum (Zic) family member transcription factor Zic1 was the most up-regulated gene in the lumbar spine (202-fold; P<10(-7)) in comparison with the iliac crest. Software analysis of differential gene expression in the biopsy samples identified the ciliary-related proteins PATCH1 and GLI-Kruppel family members Gli1 and Gli3 as part of a potential molecular network associated with Zic1. RT-PCR confirmed the expression of Zic1, Gli1, and Gli3 and other related key signaling mediators in osteoblastic cells and osteocytes in vitro. Zic1 was immunolocalized in the cytosol and nucleus of the murine osteocyte cell line MLO-Y4 and osteoblast-like cells MC3T3-E1 and in primary rat osteoblasts. MLO-Y4 cells subjected to prolonged oscillatory fluid flow showed increased localization of Zic1 in the nucleus with diminished levels in the cytosol, but no such changes were seen in MC3T3-E1 cells. A shear stress-induced increase in T-cell factor/lymphoid enhancer factor transcriptional activity was abolished by Zic1 gene silencing. These results suggest that Zic1, perhaps together with Gli1 and Gli3, may act as a link between mechanosensing and Wnt signaling. We conclude that Zic1, a neural developmental transcription factor, plays an important role in shear flow mechanotransduction in osteocytes.
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http://dx.doi.org/10.1096/fj.09-148908DOI Listing
August 2010

Striking decrease in the total precursor B-cell compartment during early childhood as evidenced by flow cytometry and gene expression changes.

Pediatr Hematol Oncol 2010 Feb;27(1):31-45

Department of Pediatrics, Faculty Division Ullevål, University of Oslo, Oslo, Norway.

The number of circulating B-cells in peripheral blood plateaus between 2 and 24 months of age, and thereafter declines gradually. How this reflects the kinetics of the precursor B-cell pool in the bone marrow is of clinical interest, but has not been studied thoroughly in humans. The authors analyzed bone marrow (n = 37) from healthy children and adults (flow cytometry) searching for age-related changes in the total precursor B-cell compartment. In an age-matched cohort (n = 25) they examined age-related global gene expression changes (Affymetrix) in unsorted bone marrow with special reference to the recombination activating gene 1, RAG1. Subsequently, they searched the entire gene set for transcripts correlating to the RAG1 profile to discover other known and possibly new precursor B-cell related transcripts. Both methods disclosed a marked, transient increase of total precursor B-cells at 6-20 months, followed by a rapid decrease confined to the first 2 years. The decline thereafter was considerably slower, but continued until adulthood. The relative composition of total precursor B-cells, however, did not change significantly with age. The authors identified 54 genes that were highly correlated to the RAG1 profile (r >or= .9, p < 1 x 10(-8)). Of these 54 genes, 15 were characteristically B-lineage associated like CD19, CD79, VPREB, EBF1, and PAX5; the remaining 39 previously not described as distinctively B-lineage related. The marked, transient increase in precursor B-cells and RAG1 transcriptional activity is not reflected by a similar peak in B-cells in peripheral blood, whereas the sustained plateau concurs in time.
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http://dx.doi.org/10.3109/08880010903420687DOI Listing
February 2010

Eight genes are highly associated with BMD variation in postmenopausal Caucasian women.

Bone 2010 Mar 14;46(3):604-12. Epub 2009 Nov 14.

Institute of Basic Medical Sciences, University of Oslo, Norway.

Low bone mineral density (BMD) is an important risk factor for skeletal fractures which occur in about 40% of women >/=50 years in the western world. We describe the transcriptional changes in 84 trans-iliacal bone biopsies associated with BMD variations in postmenopausal females (50 to 86 years), aiming to identify genetic determinants of bone structure. The women were healthy or having a primary osteopenic or osteoporotic status with or without low energy fractures. The total cohort of 91 unrelated women representing a wide range of BMDs, were consecutively registered and submitted to global gene Affymetrix microarray expression analysis or histomorphometry. Among almost 23,000 expressed transcripts, a set represented by ACSL3 (acyl-CoA synthetase long-chain family member 3), NIPSNAP3B (nipsnap homolog 3B), DLEU2 (Deleted in lymphocytic leukemia, 2), C1ORF61 (Chromosome 1 open reading frame 61), DKK1 (Dickkopf homolog 1), SOST (Sclerostin), ABCA8, (ATP-binding cassette, sub-family A, member 8), and uncharacterized (AFFX-M27830-M-at), was significantly correlated to total hip BMD (5% false discovery rate) explaining 62% of the BMD variation expressed as T-score, 53% when adjusting for the influence of age (Z-score) and 44% when further adjusting for body mass index (BMI). Only SOST was previously associated to BMD, and the majority of the genes have previously not been associated with a bone phenotype. In molecular network analyses, SOST shows a strong, positive correlation with DKK1, both being members of the Wnt signaling pathway. The results provide novel insight in the underlying biology of bone metabolism and osteoporosis which is the ultimate consequence of low BMD.
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http://dx.doi.org/10.1016/j.bone.2009.11.007DOI Listing
March 2010

Calmodulin-dependent kinase 1beta is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro.

Bone 2008 Oct 20;43(4):700-7. Epub 2008 Jun 20.

Institute of Basic Medical Sciences, Department of Biochemistry, University of Oslo, Oslo, Norway.

The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.
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http://dx.doi.org/10.1016/j.bone.2008.06.006DOI Listing
October 2008