Roser Urreizti

Effect of IL-1β, PGE2, and TGF-β1 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)‐1β, proftaglandin E2 (PGE2), and transforming growth factor‐β1 (TGF‐β1) 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‐1β treatment and decreased in response to TGF‐β1 whereas PGE2 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‐1β treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF‐β1 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. J. Cell. Biochem. 110: 304–310, 2010.

Assessment of Gene-by-Sex Interaction Effect on Bone Mineral Density

Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however, the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene‐by‐sex autosomal interactions genome‐wide, and performed expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. We conducted an autosomal genome‐wide meta‐analysis of gene‐by‐sex interaction on lumbar spine (LS) and femoral neck (FN) BMD in 25,353 individuals from 8 cohorts. In a second stage, we followed up the 12 top single‐nucleotide polymorphisms (SNPs; p < 1 × 10−5) in an additional set of 24,763 individuals. Gene‐by‐sex interaction and sex‐specific effects were examined in these 12 SNPs. We detected one novel genome‐wide significant interaction associated with LS‐BMD at the Chr3p26.1‐p25.1 locus, near the GRM7 gene (male effect = 0.02 and p = 3.0 × 10−5; female effect = −0.007 and p = 3.3 × 10−2), and 11 suggestive loci associated with either FN‐ or LS‐BMD in discovery cohorts. However, there was no evidence for genome‐wide significant (p < 5 × 10−8) gene‐by‐sex interaction in the joint analysis of discovery and replication cohorts. Despite the large collaborative effort, no genome‐wide significant evidence for gene‐by‐sex interaction was found to influence BMD variation in this screen of autosomal markers. If they exist, gene‐by‐sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP.

Mutations in the EXT1 and EXT2 genes in Spanish patients with multiple osteochondromas

Multiple osteochondromas is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours. Two causal genes have been identified, EXT1 and EXT2, which account for 65% and 30% of cases, respectively. We have undertaken a mutation analysis of the EXT1 and EXT2 genes in 39 unrelated Spanish patients, most of them with moderate phenotype, and looked for genotype-phenotype correlations. We found the mutant allele in 37 patients, 29 in EXT1 and 8 in EXT2. Five of the EXT1 mutations were deletions identified by MLPA. Two cases of mosaicism were documented. We detected a lower number of exostoses in patients with missense mutation versus other kinds of mutations. In conclusion, we found a mutation in EXT1 or in EXT2 in 95% of the Spanish patients. Eighteen of the mutations were novel.

Polymorphisms and haplotypes across the osteoprotegerin gene associated with bone mineral density and osteoporotic fractures

SummaryOsteoprotegerin plays a key role in bone remodelling. We studied the association between 24 polymorphisms and haplotypes on the OPG gene and bone mineral density and fractures. After multiple-testing correction, one SNP and two block-haplotypes were significantly associated with FN BMD. Two other block-haplotypes were associated with fracture.Introduction and HypothesisOsteoprotegerin (OPG) plays a key role in bone remodelling. Here we studied the association between polymorphisms and haplotypes on the OPG gene and bone mineral density (BMD) and fractures.MethodsTwenty-four single nucleotide polymorphisms (SNPs) were selected to cover six haplotypic blocks and were genotyped in 964 postmenopausal Spanish women. Haplotypes were established with HaploStats. Association was analysed by GLM (for BMD) and logistic regression (for fractures) both at single SNP and haplotype levels.ResultsUpon adjustment for multiple testing (p < 0.0073), one of the SNPs (SNP #17, rs1032129) remained significantly associated with FN BMD (p = 0.001). Four block-haplotypes stood multiple-testing correction. Two remained associated with FN BMD and two with fracture. The association of block-4 haplotype “AC” (of SNPs #18 and #17) with FN BMD (p = 0.0002) was stronger than that of SNP#17 alone and was the best result overall. A global assessment of the results indicated that all the alleles and haplotypes with a protective effect, at p < 0.05, belonged to a frequent long-range haplotype.ConclusionsIn conclusion, these results provide a detailed picture of the involvement of common variants and haplotypes of the OPG gene in bone phenotypes.

The p.T191M mutation of the CBS gene is highly prevalent among homocystinuric patients from Spain, Portugal and South America

In this article, one of the novel mutations, c.208_209+ 8del10, was incorrectly given as c.69_70+8del10. It corresponds to patient 64 in Table 4.

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

BackgroundMicroRNAs (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.MethodsTotal 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).ResultsBoth 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.ConclusionsWe identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy.

Molecular characterization of five patients with homocystinuria due to severe methylenetetrahydrofolate reductase deficiency

Urreizti R, Moya‐García AA, Pino‐ Ángeles A, Cozar M, Langkilde A, Fanhoe U, Esteves C, Arribas J, Vilaseca MA, Pérez‐Dueñas B, Pineda M, González V, Artuch R, Baldellou, A, Vilarinho L, Fowler B, Ribes A, Sánchez‐Jiménez F, Grinberg D, Balcells S. Molecular characterization of five patients with homocystinuria due to severe MTHFR deficiency.

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty‐two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794‐bp deletion (c.532−37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8‐bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions.Hum Mutat 32:1–8, 2011.

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.

A de novo nonsense mutation in MAGEL2 in a patient initially diagnosed as Opitz-C: similarities between Schaaf-Yang and Opitz-C syndromes

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes.