Análisis genético de enzimas de la vía esteroidal asociadas a efectos adversos musculoesqueléticos de los inhibidores de la aromatasa / Genetic analysis of steroid pathway enzymes associated with adverse musculoskeletal effects of aromatase inhibitors

Objetivos: Identificar putativas variantes funcionales en los genes CYP11A1 y CYP17A1 asociadas a efectos musculoesqueléticos (pérdida acelerada de la masa ósea y artralgias) derivados del tratamiento con inhibidores de la aromatasa (IA). Material y métodos: La cohorte B-ABLE es un estudio prospectivo de mujeres postmenopáusicas con cáncer de mama en tratamiento con IA. La densidad mineral ósea en columna lumbar y cuello femoral se midió mediante densitometría, y el dolor articular mediante escala analógica visual. A partir de polimorfismos de cambio de un nucleótido (SNPs) en los genes CYP11A1 (rs4077581, rs11632698 y rs900798) y CYP17A1 (rs4919686, rs4919683, rs4919687, rs3781287, rs10786712, rs6163, rs743572), asociados previamente con eventos musculoesqueléticos, se construyeron los haplotipos para cada paciente de la cohorte, y se seleccionaron aquellos que mostraron mayor diferencia fenotípica (p<0,05). Dentro de cada haplotipo, se eligieron aquellas pacientes con fenotipos extremos para la secuenciación de los respectivos genes y la identificación de variantes genéticas funcionales. Finalmente, se realizó un análisis de regresión lineal múltiple contemplando los modelos de herencia genética dominante, recesivo y aditivo. Resultados: No se encontró ninguna mutación en las regiones codificantes. En la región del promotor basal del gen CYP11A1 se encontró una variante genética (D15S520) asociada a la pérdida de masa ósea del cuello de fémur a los 24 meses de tratamiento con IA. Conclusiones: Variantes en regiones reguladoras del gen CYP11A1 podrían modular la expresión de este gen, explicando así parte de la variabilidad fenotípica encontrada en la pérdida de hueso de las pacientes en tratamiento con IA (AU)

Objetives: Identify putative functional variants in the CYP11A1 and CYP17A1 genes associated with musculoskeletal effects (accelerated bone mass loss and arthralgia) derived from treatment with aromatase inhibitors (AI). Material and methods: The B-ABLE cohort is a prospective study of postmenopausal women with breast cancer undergoing AI treatment. Bone mineral density in the lumbar spine and femoral neck was measured by densitometry and joint pain using visual analogue scale. From single-nucleotide polymorphisms (SNPs) in genes CYP11A1 (rs4077581, rs11632698 and rs900798) and CYP17A1 (rs4919686, rs4919683, rs4919687, rs3781287, rs10786712, rs6163, rs743572), previously associated with musculoskeletal events, haplotypes were constructed for each pacient from the cohort, and those haplotypes that showed greatest phenotypic differences were chosen (p<0.05). Within each haplotype, patients with extreme phenotypes were chosen for the sequencing of respective genes and identifying functional genetic variants. Finally, a multiple linear regression analysis was carried out considering the models of dominant, recessive and additive genetic inheritance. Results: No mutation was found in coding regions. A genetic variant (D15S520), in the basal promoter region of gene CYP11A1, was found associated with femoral neck bone loss at 24 month of AI treatment. Conclusions: Variants in regulatory regions of the CYP11A1 gene could modulate the expression of this gene, thus explaining part of the phenotypic variability found in bone loss of patients undergoing AI treatment (AU)

Superoxide dismutase downregulation in osteoarthritis progression and end-stage disease.

BACKGROUND: Oxidative stress is proposed as an important factor in osteoarthritis (OA). OBJECTIVE: To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA. METHODS: SOD expression was determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin-Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression. RESULTS: All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin-Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression. CONCLUSION: This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.

The type 2 deiodinase Thr92Ala polymorphism is associated with increased bone turnover and decreased femoral neck bone mineral density.

The role of type 2 deiodinase (D2) in the human skeleton remains unclear. The D2 polymorphism Thr92Ala has been associated with lower enzymatic activity, which could result in lower local triiodothyronine (T(3)) availability in bone. We therefore hypothesized that the D2 Thr92Ala polymorphism may influence bone mineral density (BMD) and bone turnover. We studied 154 patients (29 men, 125 women: 79 estrogen-replete, 46 estrogen-deficient) with cured differentiated thyroid carcinoma. BMD and bone turnover markers [bone-specific alkaline phosphatase (BAP), cross-linking terminal C-telopeptide of type I collagen (CTX), procollagen type 1 amino-terminal propeptide (P1NP), and cross-linked N-telopeptide of type I collagen (NTX)] were measured. Effects of the D2 Thr92Ala polymorphism on BMD and bone turnover markers were assessed by a linear regression model, with age, gender, estrogen state, body mass index (BMI), serum calcium, 25-hydroxyvitamin D, parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), and free triiodothyroxine (T(4)) as covariables. Sixty patients were wild type (Thr/Thr), 66 were heterozygous (Thr/Ala), and 28 were homozygous (Ala/Ala) for the D2 polymorphism. There were no significant differences in any covariables between the three genotypes. Subjects carrying the D2 Thr92Ala polymorphism had consistently lower femoral neck and total hip densities than wild-type subjects (p = .028), and this was accompanied by significantly higher serum P1NP and CTX and urinary NTX/creatinine levels. We conclude that in patients with cured differentiated thyroid carcinoma, the D2 Thr92Ala polymorphism is associated with a decreased femoral neck BMD and higher bone turnover independent of serum thyroid hormone levels, which points to a potential functional role for D2 in bone.

Association of aromatase and estrogen receptor gene polymorphisms with hip fractures.

UNLABELLED: Two polymorphisms of the aromatase and estrogen receptor genes appeared to interact to influence the risk of hip fractures in women. INTRODUCTION: Allelic variants of the aromatase gene have been associated with bone mineral density and vertebral fractures. Our objective was to analyze the relationship between two polymorphisms of the aromatase and estrogen receptor genes and hip fractures. METHODS: We studied 498 women with hip fractures and 356 controls. A C/G polymorphism of the aromatase gene and a T/C polymorphism of the estrogen receptor alpha gene were analyzed using Taqman assays. Aromatase gene expression was determined in 43 femoral neck samples by real-time RT-PCR. RESULTS: There were no significant differences in the overall distribution of genotypes between the fracture and control groups. However, among women with a TT genotype of the estrogen receptor, the CC aromatase genotype was more frequent in women with fractures than in controls (39 vs. 23%, p = 0.009). Thus, women homozygous for T alleles of estrogen receptor and C alleles of aromatase were at increased risk of fracture (odds ratio 2.0; 95% confidence interval 1.2-3.4). The aromatase polymorphism was associated with RNA levels in bone tissue, which were three times lower in samples with a CC genotype (p = 0.009). CONCLUSIONS: These common polymorphisms of the aromatase and estrogen receptor genes appear to interact, influencing the risk of hip fractures in women.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls.

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck's inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68-96 years). The density of eNOS+ cells (mm(-2)) was 53% lower in the inferior cortex of the fracture cases (p < 0.0004), but was similar in the superior cortex. eNOS+ osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general (p < 0.0001) and the nearest eNOS+ osteocyte was 57% further from its nearest canal surface (p < 0.0001). This differential distribution of eNOS+ osteocytes was significantly more pronounced in the cortices of fracture cases (p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS+ osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

Spatiotemporal change of rat collagenase (MMP-13) mRNA expression in the development of the rat femoral neck.

The interepiphyseal region between the greater trochanter and the capital femoral epiphysis and the medioproximal portion of the femoral neck exhibit extensive morphological changes during the first 4 weeks after birth in rats. Previous reports show that matrix metalloproteinase-13 (MMP-13, rat collagenase) mRNA is expressed in bone and cartilage during embryonal development and fracture healing. We examined MMP-13 mRNA expression and compared it with the distribution of osteopontin and osteocalcine mRNA in the femoral neck. Moreover, we examined histomorphometric analysis in the femoral neck where the morphology changes rapidly. Histomorphometric analysis of the 4-week-old rat femoral neck showed a high rate of bone formation and resorption in the region where shape changed rapidly. Osteopontin mRNA was expressed diffusely along the endosteum. In contrast, MMP-13 mRNA expression was restricted to the medial endosteal portion near the cartilage-bone interface of the femoral neck in 15- and 28-day-old rats and in the deepest endosteal interepiphyseal region of 15-day-old rats. MMP-13 mRNA-expressing osteoblastic cells were also expressing osteopontin but not osteocalcin mRNA. MMP-13 mRNA-expressing cells differ from tartrate-resistant acid phosphatase (TRAP)-positive cells, and MMP-13 mRNA-positive cells are located adjacent to TRAP-positive cells. The results of the site- and cell-specific expression of MMP-13, taken together with its enzymatic property, suggest that MMP-13 plays an important role in morphological changes in the rat femur, at least during the third and fourth week after birth, and that MMP-13 itself is involved in the interaction between osteoblastic and TRAP-positive cells.

Vitamin D concentrations in women of postmenopausal age with fractures of the femoral neck.

Thirty-eight women of postmenopausal age, suffering from fractures of the femoral neck or vertebral bodies were studied in relation to differences in bone metabolism. The blood and urinary changes concerned in mineral and bone metabolism were recorded within 10 days of trauma, and in some femoral neck fractures, a histological study of the femoral heads removed in the course of prosthetic substitution was carried out. The patients with femoral neck fractures were older than those with vertebral fractures and had metabolic and histological findings suggestive of osteomalacia. A particularly significant difference between the two groups was the plasma level of 25 hydroxycholecalciferol, which was lower in femoral neck fractures, and the urinary excretion of calcium, which was also more reduced in femoral neck fractures. A deficit of vitamin D hepatic metabolite thus appears to be a risk factor for femoral neck fractures in old patients.