Angela Xuereb-Anastasi

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.

Associations of polymorphisms in the vitamin D receptor gene (BsmI and FokI) with bone mineral density in postmenopausal women in Malta

Previous studies have suggested that variations in the vitamin D receptor (VDR) gene are related to bone mineral density (BMD). In this study, the T→C transition in the start codon and the G→A polymorphism at the 3′ end of the VDR gene, identified by endonucleases FokI and BsmI, respectively, were analysed and correlated with BMD in postmenopausal Maltese women (n=104). Genotype frequencies observed for the VDR start codon polymorphism (SCP) were CC: 60.4%; CT: 30.7% and TT: 8.9%, while those observed for the 3′ in this study were GG: 16.4%; GA: 51.9%; AA: 31.7%. In postmenopausal women, both lumbar and femoral BMD were observed to be highest in CC homozygotes for the FokI genotype and in GG homozygotes for the BsmI genotype, although in both groups the difference between the genotypes was not statistically significant, even after adjusting BMD for age, BMI and years since menopause. No evidence of linkage disequilibrium between the two alleles was observed.

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 β-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. β-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 β-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.

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.

Variants within protectin (CD59) and CD44 genes linked to an inherited haplotype in a family with coeliac disease

Coeliac disease (CD) is an autoimmune disorder characterised by inflammation, villous atrophy and hyperplasia of the small intestinal mucosa that affects genetically susceptible individuals. A genome-wide scan was performed in 17 family members with high incidence of CD. Highest nonparametric linkage (NPL) and logarithm of odds (LOD) scores were of 6.21 (P = 0.0107) and 2.57, respectively, to a region on chromosome 11p13-12. Following fine mapping, NPL and LOD scores did not change, but the linkage interval on chromosome 11 was narrowed to a region that is approximately 50.94 cM from pTer. Two inherited haplotypes on chromosomes 11p13-12 and 9q21 were observed in all affected members but not in the majority of clinically normal individuals. Sequencing of genes at region 11p13-12 showed a number of sequence variants, two of which were linked with the inherited haplotype. One of these variants in the CD59 gene was found at a very low frequency in the population and could possibly affect pre-messenger RNA splicing. This study is of particular importance for the identification of novel genes that might be responsible for CD other than human leukocyte antigen.

Effects of SNPs in the Col1a1 and Methylenetetrahydrofolate Reductase Genes on BMD in Postmenopausal Women in Malta

Effects of SNPs in the Col1a1 and Methylenetetrahydrofolate Reductase Genes on BMD in Postmenopausal Women in Malta 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), Wards 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 C allele 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.

Frequency of the CCR5‐Δ32 polymorphism in the Maltese population at birth

In this study, the frequency of the CCR5‐Δ32 polymorphism was estimated in the human population of Malta. The frequency of the CCR5‐Δ32 allele was found to be 1.1% which was similar to that of other island populations, and agree with the north to south gradient observed across Europe.

Biochemical Predictors of Bone Mineral Density and Fracture Susceptibility: Results from a Maltese Study

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 Osteoporosis [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].

No effects of a synonymous variant within the CD59 gene on its protein product in duodenal biopsies of coeliac individuals

A novel rare variant within the CD59 gene was linked with coeliac disease in a family with high incidence of disease. Functional analyses of this variant were performed using complementary DNA analysis and protein analysis in paraffin-embedded duodenal biopsies from affected individuals and controls. No effects on pre-mRNA or size of linear protein were observed, although these results do not exclude the possible effects of this variant on co-translational protein folding.