Search

BACKGROUND/INTRODUCTION: Epidemiological studies and translational models have highlighted the importance of WNT16 as a key regulator of bone mineral density (BMD)., PURPOSE: The study aimed to investigate the effect of two WNT16 variants, a frameshift rs55710688 (insCCCA) in the Kozak sequence, and a single nucleotide variant rs3801387 (A>G) located in the last intron of WNT16, with BMD and fracture risk in the Maltese postmenopausal women., METHODS: Genotyping was performed in 1,045 women from the Malta Osteoporotic Fracture Study using Competitive Allele Specific PCR (rs55710688) and TaqMan® fluorogenic 5’ nuclease allelic discrimination (rs3801387). Genotype-phenotype associations were analysed using the Mann-Whitney statistic whereas odds ratios (OR) with 95% confidence intervals [CI] were computed by logistic regression analysis adjusted for confounders., RESULTS: Genotyping of the WNT16 rs55710688 and rs3801387 was successful in 1,038 (CCCA=24%) and 1,027 (G=27%) samples respectively. Women with the homozygous reference genotype for both WNT16 variants had a lower lumbar spine (LS) T-score relative to women with the homozygous alternative genotype (rs55710688 p=0.035; rs3801387 p=0.031). Risk ratios revealed that homozygosity for the reference alleles was associated with osteoporosis at the LS (rs55710688 adjusted-OR: 2.44 [1.16-5.13]; rs3801387: 2.40 [1.18-4.89]), and all-type of low-trauma fracture risk which was not attenuated by BMD (rs55710688: 2.17 [1.11-4.27]; rs3801387: 1.90 [1.05-3.55]). WNT16 rs55710688 reference genotype also exhibited a deleterious effect on femoral neck BMD (3.10 [1.05-9.15]). Finally, the haplotype with the reference alleles for WNT16 rs55710688 and rs3801387 was associated with LS BMD (p=0.007) and fracture risk (p=0.021)., CONCLUSION(S): Results indicate that the WNT16 rs55710688 and rs3801387 variants are possible genetic determinants of site-specific BMD and fracture risk in Malta, which is in line with other epidemiological studies. Our findings support the results of in vitro assays and in silico modelling demonstrating reduced translational efficiency in the presence of the reference alleles culminating in lower bone formation., peer-reviewed

BACKGROUND & OBJECTIVES: Runt-related transcription factor 2 (RUNX2) is a major transcription factor involved in osteoblast and chondrocyte differentiation, skeletogenesis and fracture repair. Transactivation of RUNX2 is under tight regulatory control particularly via promoter 2 (P2). The study aimed to assess the effect of the P2 RUNX2 T-1025C variant in relation to bone mineral density (BMD) at different anatomical sites, fracture risk and levels of biochemical parameters in the Maltese population., METHODS: Genotyping was performed in 1,045 Maltese postmenopausal women from the Malta Osteoporotic Fracture Study using the TaqMan® fluoregenic 5' nuclease allelic discrimination assay. Genotype-phenotype associations were analysed using the Mann-Whitney statistic whereas odds ratios with 95% confidence intervals were computed using logistic regression analysis adjusted for confounders., RESULTS: Genotyping was successful in 1,043 samples, with the reference T and alternative C alleles observed at a frequency of 0.92 and 0.08 respectively. Women aged >60 years with the TC genotype had higher total serum calcium (p=0.029) and lower total serum alkaline phosphatase (ALP) levels (p=0.046) relative to women with the TT genotype. Additionally, carriers of the C allele showed higher femoral neck BMD than homozygous carriers of the T allele. Nonetheless, the latter did not reach statistical significance (p>0.05). Homozygosity for the C allele was associated with an almost 5-fold increased fracture risk compared to homozygosity for the T allele, which was not attenuated after adjusting for BMD (adjusted OR: 4.9 [1.2-19.6], p=0.025). This is the first study to report an association with fractures. No association was seen with lumbar spine or total hip BMD., CONCLUSION: Results indicate that the RUNX2 T-1025C is a possible genetic determinant of fracture risk in the Maltese population, as well as calcium and ALP control. This functional variant alters the binding of several transcriptional activators and repressors, possibly affecting bone composition and strength., peer-reviewed

INTRODUCTION: Osteoporosis is a complex metabolic and skeletal disease having a strong genetic background. Indeed, heritability of bone mineral density (BMD) in twin and family studies ranges from 50 to 85%. The aim of the study was to identify known and/or novel genes and gene variants that play a role in the susceptibility of primary osteoporosis in an extended Maltese family., MATERIALS AND METHODS: A 2-generation family having multiple relatives with osteoporosis (T-score, RESULTS: Eleven shortlisted variants segregating in a dominant inheritance pattern were identified in the affected relatives having a minor allele frequency of ≤2%. Variants included missense variants within ADAMTS20 (rs138035327), ARSD (rs78034736), BMP1 (rs368615556), CLDN18 (rs114998965), SELP (rs754086574), TGFβ2 (rs773943154), TRIM45 (rs146244405), PCDHGA11 (rs138408376), PLEC (rs138924815) and SPARC (rs41290587), and one stop gain variant within WDR89 (rs944955056)., CONCLUSIONS: Future studies will evaluate the shortlisted variants by replicating in the Malta Osteoporotic Fracture Study - a case-control collection of more than 1000 Maltese postmenopausal women and other extended Maltese pedigrees so as to determine association with osteoporosis and low-trauma fracture risk at different anatomical sites. Top candidates will in turn be assessed using functional studies., peer-reviewed

INTRODUCTION: Osteoporosis is a skeletal disease with a strong genetic basis. A study on an extended Maltese family with a highly penetrant form of osteoporosis, revealed the presence of the rs146089604 variant (c.686+32G>A) in intron 7 of the Zinc finger protein 384 (ZNF384) gene, predicted to affect pre-messenger RNA (mRNA) splicing. The aim of this study was to assess the functional effect of the variant using an exon-trapping vector transfected in three human cell types., METHODS: The target DNA region harbouring G or A allele was inserted in the p.SPL3 vector, creating mini-gene constructs that were transfected in human kidney-derived cells (HEK-293) and two human osteoblasts-derived cells (SaOS-2 and h-FOB). Extracted mRNA was converted into complementary DNA (cDNA), amplified by PCR and sequenced to determine the transcript size and identify any splicing variants., RESULTS: Mini-gene construct with the alternative A allele lead to exon 8 and part of intron 8 to be retained, both of which were spliced off in the presence of the G allele. These results were observed for constructs transfected in the osteoblasts-derived cell lines. In HEK-293 cells, no difference in transcript size was seen for the G or A allele, suggesting different splicing mechanisms., CONCLUSION: Observations may indicate that the ZNF384 rs146089604 could be a causal variant contributing to osteoporosis. ZNF384 transactivates type I collagen and matrix metalloproteinases, and suppresses bone morphogenic protein (BMP) and Wnt signalling resulting in reduced bone volume and strength. Thus, impaired ZNF384 splicing could alter the protein’s function affecting bone homeostasis., peer-reviewed

INTRODUCTION: Osteoporosis is a complex metabolic bone disease having a strong genetic background and high heritability rate. An extended Maltese family having multiple members affected with primary osteoporosis (T-score, METHODS: A 2-generation family consisting of 15 relatives with ages ranging from 28 to 74 years was recruited. Biochemical analysis excluded any comorbidities affecting bone health and none of the relatives had osteogenesis imperfecta. Whole genome sequencing was performed on 12 relatives and a number of filtering schemes together with in silico modelling were applied to narrow down the list of potentially causal variants., RESULTS: Five missense variants segregating in a dominant inheritance pattern were shortlisted, all of which had an alternative allele frequency of ≤1% in the 1000Genome project. The gene variants identified were ADAMTS20 rs138035327, BMP1 rs368615556, SELP rs754086574, TGF-b2 rs773943154 and TRIM45 rs146244405. Replication of the ADAMTS20 rs138035327 variant in a case-control collection of 1045 Maltese postmenopausal women was performed to determine association with bone mineral density, fragility fracture risk and biochemical parameters, including serum calcium and alkaline phosphatase. Heterozygosity was associated with a 2-fold increased risk of low serum calcium levels (odds ratio: 2.3 (95% confidence interval 1.1-5.0), p=0.03)., CONCLUSION: ADAMTS20 encodes a protease enzyme that cleaves aggrecan, required for cartilage and bone formation. Thus, functional follow-up is required to determine how the ADAMTS20 variant could be affecting calcium and bone homeostasis., DISCLOSURES: This work was funded by the Endeavour Scholarships Scheme., peer-reviewed

Osteoporosis is a multifactorial skeletal disease characterised by low bone mass and micro architectural deterioration, leading to increased fracture susceptibility [1,2]. In Malta, 20% of women and 6% of men aged 50 years and older are estimated to be affected with steoporosis [3]. Fracture is the most significant clinical consequence of osteoporosis, with the most common, debilitating, and costly fractures being those of the spine, hip and wrist [1]. A number of environmental and genetic risk factors are known to affect bone mineral density (BMD), which in turn impacts fracture outcome [4,5]. Furthermore, other parameters reflecting calcium homeostasis, matrix mineralisation, and bone formation can also be targeted and measured in blood. Levels of serum calcium, serum albumin, and total serum alkaline phosphatase (sALP) are suggested as potential indicative markers of osteoporosis and/or fracture susceptibility, and increased frailty [5,6]., peer-reviewed

BACKGROUND: Osteoporosis and fracture risk are polygenic conditions which result from an interplay of genetic, biochemical and environmental factors. A number of biochemical markers including serum calcium, total alkaline phosphatase (ALP) and albumin, were analysed in relation to bone mineral density (BMD) and different types of low-trauma fractures in Maltese postmenopausal women. Levels were also correlated with a number of clinical risk factors including physical activity and years since menopause (YSM)., METHODS: An age-matched case-control study of 1045 women was performed. Women who suffered low-trauma fractures were classified as cases whereas those without a fracture history were included as controls subdivided into normal, osteopenic or osteoporotic according to their BMD status. Blood specimens were collected following good standard practice and within 18 hours of fracture in the case of fresh-trauma fractures. Biochemical testing was performed using spectrophotometric analysis., [excerpt], peer-reviewed

BACKGROUND: Osteoporosis is a multifactorial skeletal disease characterised by low bone mass leading to increased fracture risk. Members of the low-density lipoprotein receptor-related protein (LRP) gene family play a role in osteoblastogenesis through the Wingless (Wnt)/Beta-catenin pathway. LRP4 con- trols the actions of sclerostin, a Wnt inhibitor, whereas LRP5 promotes bone formation. The aim was to evaluate the effect of four non-synonymous coding polymorphisms in relation to bone mineral density (BMD) and low-trauma fractures in Mal- tese postmenopausal women. Genotyped variants were the LRP4 rs2306033 (C>T) and rs6485702 (G>A) variants, and the LRP5 rs4988321 (G>A) and rs3736228 (C>T) variants., METHODS: Research subjects were 1045 women aged 40 to 79 years, subdivided in three BMD groups without a fracture history: normal, osteopenic or osteoporotic. Women with a fracture history were classified as cases. Genotyping was performed by polymerase chain reaction and restriction frag- ment length polymorphism, or by real-time PCR. Odds ratios were computed using logistic regression analysis adjusted for age and clinical risk factors., [excerpt], peer-reviewed

INTRODUCTION: Osteoporosis and fractures are complex skeletal conditions resulting from an interplay of genetic and environmental factors. The aim of the study was to investigate the association of biochemical levels of total serum calcium, total serum alkaline phosphatase (sALP) and serum albumin with bone mineral density (BMD) levels at the lumbar spine (LS) and femoral neck (FN), and with fracture risk in Maltese postmenopausal women. Levels were also correlated with age, years since menopause (YSM) and physical activity., METHODS: A case-control study of 1045 women was performed. Women who suffered a fracture were classified as cases whereas women without a fracture history were included as controls subdivided into normal, osteopenic or osteoporotic according to their BMD measurements. Blood specimens were collected following good standard practice and testing was performed by spectrophotometry., RESULTS: Calcium, and to a lower extent sALP, were correlated with FN BMD levels. Fracture cases, especially those who sustained a hip fracture, had the lowest levels of calcium, sALP and albumin relative to all other control groups. Biochemical levels decreased with increasing age, possibly increasing fracture risk. YSM was correlated with lower calcium levels in fracture cases (rho: 0.229, P, CONCLUSION: Results suggest that levels of calcium, sALP and albumin could be indicative of fracture risk, whereas calcium levels and to lower extent sALP are indicators of hip BMD., peer-reviewed

BACKGROUND: Osteoporosis is a hereditary multifactorial disease characterised by low bone mass leading to an increased susceptibility to fracture. Bone mineral density (BMD) is the most widely used predictor of fracture risk. Gene variants have been found associated with a low BMD and increased fracture risk; nonetheless studies have identified the relationship between susceptibility genes and fractures independent of BMD., OBJECTIVE: Eight single nucleotide polymorphisms (SNPs) within four candidate genes were investigated for their effect on BMD at different anatomical sites and with different low-trauma fractures., METHODS: One thousand and forty-five maltese postmenopausal women were recruited and BMD measurements were performed by dual-energy X-ray absorptiometry. Women who suffered low-trauma fractures were classified as cases whereas subjects without a fracture history were included as controls. Informed consent was obtained from all participants. Genotyping was performed by PCR followed by restriction fragment length polymorphism, and RT PCR high resolution melt., RESULTS: Using logistic regression analysis adjusted for age, three SNPs in three genes (LRP5 (rs3736228), RANK (rs3018362) and OPG (rs2073618)) were found associated with a low BMD and increased risk of all-type of low trauma fractures (P, CONCLUSION: Results from this independent replication study indicate that a number of gene variants are associated with reduced BMD and/or increased fracture susceptibility in Maltese postmenopausal women., peer-reviewed

INTRODUCTION: The Low-density lipoprotein receptor-related protein 5 (LRP5) is involved in osteoblast differentiation, making it an important determinant of bone mass and strength. The single nucleotide polymorphism, SNP (C>T) at position 1330 in exon 18 results in a missense substitution of alanine to valine (A1330V) which has been associated with low bone mineral density (BMD) and increased fracture risk., AIM: To investigate the influence of the A1330V polymorphism on BMD and different low-trauma fractures in Maltese postmenopausal women., METHODOLOGY: 1043 women between 40 and 79 years were recruited and their BMD measured by dual-energy X-ray absorptiometry. Subjects without a history of a fragility fracture were subdivided in three groups: normal (n=223), osteopenic (n=271), and osteoporotic (n=282) according to their BMD. The remaining 267 were fracture cases who had a normal (n=12), osteopenic (n=107) or osteoporotic BMD (n=148). Genotyping was performed by polymerase-chain reaction and restriction enzyme digest., RESULTS: The genotype distributions were as follows: normal controls CC (78%), CT (21%), TT(1%); osteopenic subjects CC (78%), CT (20%), TT (2%); osteoporotic subjects CC (68%), CT (28%), TT (4%); and fracture cases CC (69%), CT (27%), TT (4%). In the total study group, the A1330V SNP was associated with reduced lumbar spine BMD (TT: age-adjusted Odds ratio, OR 3.7 [95% Confidence Interval 1.2-11.0], p=0.02; CT: OR 1.6 [1.1-2.4], p=0.01) and to a lesser extent reduced femoral neck BMD (TT: OR 2.8 [0.9-8.7], p=0.07; CT: OR 1.7 [1.2-2.6], p=0.01). Women without a fracture history had an increased risk of osteoporosis when carrying one or both copies of the minor allele T (CT: OR 1.6 [1.0-2.4], p=0.04; TT: OR 10.6 [1.4-80.5], p=0.03). No significant difference was observed between osteopenic subjects (without fractures) and normal controls. When comparing fracture cases to normal controls, women carrying CT/TT genotypes had an increased fracture risk than women with the CC genotype (CT: 1.6 [1.0-2.4], p=0.05; TT: OR of 8.3 [1.0-67.0], p=0.05). All fracture cases homozygous for the T allele had an osteopenic or osteoporotic BMD; fracture risk was partly attenuated by BMD adjustment (TT: OR 2.5 [0.2-27.7], p=0.45) and remained unchanged in carriers of the A1330V SNP (CT: OR 1.4 [0.7-2.8] p=0.31). The TT genotype was the most common among subjects with a wrist, humerus or hip fracture; however the difference was not significant (p>0.05)., CONCLUSION: The results indicate that the A1330V polymorphism is associated with reduced BMD and increased fracture susceptibility in Maltese postmenopausal women., peer-reviewed

Recently the Aryl hydrocarbon receptor – interacting protein (AIP) gene attracted attention as a novel gene linked to familial cases of acromegaly. In Malta the predicted prevalence of pituitary adenomas is particularly high, 4.67 per 10,000 population, thus suggesting a genetic predisposition. Fourty seven maltese patients with acromegaly were screened for germ-line mutations in the AIP gene. Pituitary tumour tissue, removed during transphenoidal surgery from eight patients was cultured using a locally optimized protocol. Functional analysis of wild-type and mutant AIP genes on cell survival and proliferation of these primary cell cultures was carried out by transfection and proliferation assays (MTT). Two variants of the AIP gene, the R304X mutant (shown to generate a non-functional truncated protein, and R9Q, the mutant identified in a Maltese patient suffering from acromegaly (Farrugia et al. 2008, unpublished data), were successfully transfected into the primary cells. Transfection of wild-type, R304X and R9Q variants demonstrated that the R304X variant loses the ability to reduce proliferation as compared to the wild-type AIP, an effect previously demonstrated in other cell lines but not in primary cells. Furthermore, the R9Q mutation shows an inverse behaviour, causing a significant increase in proliferation in the primary cells, hence pointing to a gain-of-function mutation in the AIP gene. AIP protein has been postulated to interact with the cAMP pathway and cell cycle regulators. Our results provide evidence supporting a role for AIP as a tumour suppressor gene. The R9Q mutant could help clarify the role of the N-terminal of the gene, which at present remains speculative Ongoing studies utilizing immunohistochemistry, cAMP assays and q-PCR should help identify AIP alterations downstream effects and verify whether specific AIP variants may alter cAMP levels and regulate gene expression of regulators and transcription factors., peer-reviewed

Osteoporosis is a complex metabolic disease with a strong genetic component which results in a decrease in bone mineral density and deterioration in the microarchitecture of bone, leading to an increased risk of fractures. During the last decade, a number of genetic studies of the Maltese population were performed to determine a potential genetic component which increases the individual’s susceptibility to osteoporosis. Both family and population case-control approaches were used. Linkage analysis using large affected families has identified a region on chromosome 11p12, following which functional studies have shown that variations within two genes found in that region namely, TRAF6 and CD44, affect gene expression, and, more specifically, pre-mRNA splicing in the case of CD44. This is the first report of strong suggestive linkage of 11p12 to osteoporosis. Using the candidate gene approach, selected candidate genes, which had been studied in other populations and been found to increase susceptibility to osteoporosis to varying degrees in some of them, included those coding for the vitamin D and oestrogen receptors (VDR & ER1), osteoprotegerin (TNFRSF11B), collagen type 1 (COL1A1), methylenetetrahydrofolate reductase (MTHFR) and lipoprotein receptor related protein (LRP)-5. In the Maltese population, the most significant result showed that a polymorphism in the promoter region of TNFRSF11B, which codes for osteoprotegerin, had a significant effect on bone mineral density, thus increasing the susceptibility to osteoporosis. These studies have shown that variations within three different genes, namely TRAF6, CD44 and TNFRSF11B, all involved in osteoclast biology, might increase the risk of osteoporosis and could therefore serve as targets for novel therapeutic agents., peer-reviewed

Two common single nucleotide polymorphisms (SNPs) within the COL1A1 gene and the C677T variant within the methylenetetrahydrofolate reductase (MTHFR) gene have been studied for correlation with bone mineral density (BMD) in 126 postmenopausal Maltese women (55.6 ± 7.1 years). All polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while BMD at the lumbar spine (LS), femoral neck (FN), Ward’s triangle and trochanter was measured by dual energy X-ray absorptiometry (DEXA). The observed genotype frequencies were similar to those in other populations and were in Hardy-Weinberg equilibrium. No association was observed between polymorphisms in the COL1A1 gene and BMD, even after adjustment for age, body mass index (BMI) and years since menopause. The Callele of the C677T variant of the MTHFR gene had a negative effect on trochanter BMD when testing for genetic models of dominant and recessive alleles (independent sample t-test: p = 0.03). Genotype frequencies of both genes did not differ significantly between normal women and those with a low BMD at either the LS or FN., N/A