Whole Genome Sequencing Unravels New Genetic Determinants of Early-Onset Familial Osteoporosis and Low BMD in Malta.

BACKGROUND: Osteoporosis is a skeletal disease with a strong genetic background. The study aimed to identify the genetic determinants of early-onset familial osteoporosis and low bone mineral density (BMD) in a two-generation Maltese family. METHODS: Fifteen relatives aged between 28-74 years were recruited. Whole genome sequencing was conducted on 12 relatives and shortlisted variants were genotyped in the Malta Osteoporotic Fracture Study (MOFS) for replication. RESULTS: Sequential variant filtering following a dominant inheritance pattern identified rare missense variants within SELP, TGF-ß2 and ADAMTS20, all of which were predicted to be likely pathogenic and participate in osteoimmunology. TGF-ß2 c.1136C>T was identified in five individuals from the MOFS in heterozygosity, four of whom had osteopenia/osteoporosis at the lumbar spine and hip, and/or had sustained a low-trauma fracture. Heterozygosity for the ADAMTS20 c.4090A>T was accompanied by lower total hip BMD (p = 0.018) and lower total serum calcium levels in MOFS (p < 0.01), recapitulating the findings from the family. Women carrying at least one copy of the alternative allele (TC/CC) for SELP c.2177T>C exhibited a tendency for lower lumbar spine BMD and/or wrist fracture history relative to women with TT genotype. CONCLUSIONS: Our findings suggest that the identified variants, alone or in combination, could be causal factors of familial osteoporosis and low BMD, requiring replication in larger collections.

Pharmacogenomic Effects of ß-Blocker Use on Femoral Neck Bone Mineral Density.

CONTEXT: Recent studies have shown that ß-blocker (BB) users have a decreased risk of fracture and higher bone mineral density (BMD) compared to nonusers, likely due to the suppression of adrenergic signaling in osteoblasts, leading to increased BMD. There is also variability in the effect size of BB use on BMD in humans, which may be due to pharmacogenomic effects. OBJECTIVE: To investigate potential single-nucleotide variations (SNVs) associated with the effect of BB use on femoral neck BMD, we performed a cross-sectional analysis using clinical data, dual-energy x-ray absorptiometry, and genetic data from the Framingham Heart Study's (FHS) Offspring Cohort. We then sought to validate our top 4 genetic findings using data from the Rotterdam Study, the BPROOF Study, the Malta Osteoporosis Fracture Study (MOFS), and the Hertfordshire Cohort Study. METHODS: We used sex-stratified linear mixed models to determine SNVs that had a significant interaction effect with BB use on femoral neck (FN) BMD across 11 gene regions. We also evaluated the association of our top SNVs from the FHS with microRNA (miRNA) expression in blood and identified potential miRNA-mediated mechanisms by which these SNVs may affect FN BMD. RESULTS: One variation (rs11124190 in HDAC4) was validated in females using data from the Rotterdam Study, while another (rs12414657 in ADRB1) was validated in females using data from the MOFS. We performed an exploratory meta-analysis of all 5 studies for these variations, which further validated our findings. CONCLUSION: This analysis provides a starting point for investigating the pharmacogenomic effects of BB use on BMD measures.

Biochemical Predictors of Low Bone Mineral Density and Fracture Susceptibility in Maltese Postmenopausal Women.

Osteoporosis and fractures are complex conditions influenced by an interplay of genetic and environmental factors. The aim of the study was to investigate three biochemical parameters including total serum calcium, total serum alkaline phosphatase (sALP) and albumin in relation to bone mineral density (BMD) at the lumbar spine and femoral neck (FN), and with all-type of low-trauma fractures in Maltese postmenopausal women. Levels were also correlated with age and physical activity. 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. Calcium and sALP levels were weakly correlated with FN BMD levels (calcium: r = -0.111, p = 0.002; sALP: r = 0.089, p = 0.013). Fracture cases had the lowest serum levels of calcium, sALP and albumin relative to all other control groups, which decreased with increasing age, possibly increasing fracture risk. Biochemical levels were lowest in women who sustained a hip fracture and more than one fracture. Biochemical parameters decreased with reduced physical activity; however, this was most evident for fracture cases. Reduced physical activity was associated with lower BMD levels at the hip, and to a lower extent at the spine. In conclusion, results suggest that levels of serum calcium and albumin could be indicative of fracture risk, whereas calcium levels and to lower extent sALP levels could be indicators of hip BMD.

Expression and clinical significance of Wnt players and survivin in pituitary tumours.

Deregulation of the Wnt pathway has been implicated in oncogenesis of numerous tissues including the pituitary gland. Immunohistochemical localization and quantification of ß-catenin, Cyclin D1, c-MYC and Survivin expression in 47 pituitary adenomas (35 non-functioning, seven GH-secreting, three prolactinomas, two ACTH-secreting tumour) and six normal controls was undertaken in this study and correlation of protein expression to patient and tumour characteristics analysed. ß-catenin was strictly membrane-bound with no difference observed between normal and tumour tissue. In contrast, Cyclin D1 and c-MYC localization was nuclear and significantly higher in tumour versus normal tissue (p < 0.05). c-MYC expression correlated negatively with age at diagnosis (p = 0.006, R = -0.395) while Cyclin D1 expression correlated positively with age (p = 0.036, R = 0.306) and was higher in males than in females (p = 0.036). c-MYC expression was significantly lower in patients with functional tumours requiring octreotide treatment and in patients with non-functioning tumours suffering from hypopituitarism. Survivin expression was extremely low in tumours and absent in normal controls. Involvement of the canonical Wnt pathway appears to be minimal, given the segregation of ß-catenin to the membrane. Our data suggest that c-MYC may have an important role in early pituitary tumorigenesis while Cyclin D1 is likely to promote tumour growth at a later stage. We also report a novel gender difference in Cyclin D1 expression, the biological significance of which merits further analysis. The reported reduction of c-MYC in functional tumours subsequently treated with octreotide further supports a role of c-MYC in early tumorigenesis and not in recurrence. The decrease in c-MYC in patients with hypopituitarism provides the first in vivo evidence for hormonal regulation of c-MYC expression.

Functional polymorphisms within the TNFRSF11B (osteoprotegerin) gene increase the risk for low bone mineral density.

Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. Osteoprotegerin (OPG), the product of this gene, is a key negative regulator of osteoclastogenesis and is secreted by osteoblasts/stromal cells. A previous study in Maltese postmenopausal women showed positive association of low bone mineral density (BMD) with a polymorphism found within the promoter region of this gene (C950T). In this study, direct DNA sequencing revealed 12 variants with polymorphisms C950T, G1181C and rs4876869 observed to be in strong linkage disequilibrium. The constructed haplotype T-G-T was found to increase the risk for a low BMD, while C-G-T and C-C-C have a protective role; thus, we investigated the functional role of both C950T and rs4876869 in vitro. The promoter region, including the C950T alleles, was amplified by PCR, cloned into pGL3 enhancer vector and transfected into HeLa, COS-7 and RAW264.7 cell lines. After incubation, luciferase activity was measured. The T/C (rs4876869) change was tested for its possible effect on pre-mRNA splicing, using an exon-trapping vector. A statistical significant difference in gene expression was observed between the alleles for T950C, with the T allele showing a lower luciferase expression in all cell lines (P<0.01). For rs4876869, exon skipping was observed for the C allele, while only one transcript harbouring the whole exon was observed for the T allele. Our findings suggest that the T-G-T haplotype might be increasing the risk for osteoporosis due to lower quantities of the full OPG transcript being expressed resulting in a higher bone resorption.

Effects of a synonymous variant in exon 9 of the CD44 gene on pre-mRNA splicing in a family with osteoporosis.

In a previous linkage study, suggestive linkage to osteoporosis was observed in marker D11S1392 on chromosome 11p12. The CD44 gene, found at this locus, was sequenced in one of the families studied. Sequencing all coding regions and promoter in affected and non-affected family members revealed a number of sequence variants, one of which was found to be linked and inherited identical by descent together with the linked STR allele. This G to A variant, which does not cause an amino acid change, was found in exon 9 of the CD44 gene, 32 base pairs upstream from the exon-intron junction. Preliminary analysis using a bioinformatics tool suggested that the presence of the A allele abolished an exon splicing enhancer (ESE) site, thus possibly affecting RNA splicing. It was observed using an exon-trapping vector, that in the presence of the A allele, only one transcript was observed in RAW264.7 cells, as opposed to two transcripts transcribed in the presence of the G allele. These observations suggest that the linked synonymous variant found in exon 9 of the CD44 gene might be increasing susceptibility to osteoporosis in this family by affecting the splicing mechanism.