Omar M.E. Albagha

Promoter and intron 1 polymorphisms of COL1A1 interact to regulate transcription and susceptibility to osteoporosis

Three polymorphisms (-1997G/T; -1663IndelT and +1245G/T) have been identified in the 5 flank of COL1A1 gene that are associated with osteoporosis but the underlying mechanism is unclear. Here we investigated the functional effects of these variants on COL1A1 transcription. Transcription was 2-fold higher with the osteoporosis-associated G-del-T haplotype compared with the common G-Ins-G haplotype. Gel shift assays showed that the region surrounding the -1663IndelT polymorphism recognized a complex of proteins essential for osteoblast differentiation and function including Nmp4 and Osterix, and the osteoporosis-associated -1663delT allele had increased binding affinity for this complex. Chromatin immunoprecipitation assays confirmed that the region flanking -1663insdelT bound a complex of proteins including Osterix and Nmp4 and also showed evidence of recruitment of Nmp4 to the Sp1 binding site in intron 1. Further studies showed that haplotype G-del-T had higher binding affinity for RNA polymerase II, consistent with increased transcription of the G-del-T allele and there was a significant inverse association between carriage of G-del-T and bone mineral density (BMD) in a cohort of 3270 Caucasian women. We conclude that common polymorphic variants in the 5 flank of COLIA1 regulate transcription by affecting DNA-protein interactions and that increased levels of transcription correlate with reduced BMD values in vivo. This is consistent with a model whereby increased COL1A1 transcription predisposes to osteoporosis, probably by increasing production of the alpha 1 chain and disrupting the normal ratio of collagen type 1 alpha 1 and alpha 2 chains.

Phenotype presentation of hypophosphatemic rickets in adults.

Hypophosphatemic rickets (HR) is a group of rare disorders caused by excessive renal phosphate wasting. The purpose of this cross-sectional study of 38 HR patients was to characterize the phenotype of adult HR patients. Moreover, skeletal and endodontic severity scores were defined to assess possible gender differences in disease severity in patients with genetically verified X-linked HR. Compared to normal reference data, i.e., zxa0=xa00, HR patients had significantly lower final height, with a mean difference in z-score of −1.9 (95% CI −2.4 to −1.4, Pxa0<xa00.001). Compared to paired z-scores of final height, z-scores of leg length were significantly lower and those of sitting height were significantly higher (Pxa0<xa00.001), resulting in disproportion as indicated by the significantly elevated sitting height ratio, mean difference in z-score of 2.6 (95% CI 2.1–3.1, Pxa0<xa00.001). Z-scores of head circumference (median 1.4, range −0.4 to 5.5, Pxa0<xa00.001) and z-scores of bone mineral density (BMD) of the lumbar spine (median 1.9, range −1.5 to 8.6, Pxa0<xa00.001) were significantly elevated compared to normal reference data. The relative risk (RR) of fracture was reduced (RRxa0=xa00.34, 95% CI 0.20–0.57, Pxa0<xa00.001). The skeletal severity score tended to be higher in males compared to females (Pxa0=xa00.07), and no gender difference in endodontic severity was found. In conclusion, adult HR patients were characterized by short stature and were disproportioned. They had elevated BMD of the lumbar spine and a reduced risk of fractures. We found a tendency for males to be more severely affected than females.

Genetics of Paget’s Disease of Bone

Paget’s disease of bone (PDB) is a common condition, which is characterised by focal areas of increased and disorganized bone remodeling. Genetic factors play an important role in the disease. In some cases, Paget’s disease is inherited in an autosomal dominant manner and the most common cause for this is a mutation in the SQSTM1 gene. Other familial cases have been linked to the OPTN locus on Chromosome 10p13 and still other variants have been identified by genome wide association studies that lie within or close to genes that play roles in osteoclast differentiation and function. Mutations in TNFRSF11A, TNFRSF11B and VCP have been identified in rare syndromes with PDB-like features. These advances have improved understanding of bone biology and the causes of PDB. The identification of genetic markers for PDB also raises the prospect that genetic profiling could identify patients at high risk of developing complications, permitting enhanced surveillance and early therapeutic intervention.

Novel KRAS Gene Mutations in Sporadic Colorectal Cancer

Introduction In this article, we report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi sporadic colorectal cancer patients from the Eastern Province. Methods Genomic DNA was extracted from formalin-fixed, paraffin-embedded cancerous and noncancerous colorectal tissues. Successful and specific PCR products were then bi-directionally sequenced to detect exon 4 mutations while Mutector II Detection Kits were used for identifying mutations in codons 12, 13 and 61. The functional impact of the novel mutations was assessed using bioinformatics tools and molecular modeling. Results KRAS gene mutations were detected in the cancer tissue of 24 cases (42.85%). Of these, 11 had exon 4 mutations (19.64%). They harbored 8 different mutations all of which except two altered the KRAS protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. One mutation is predicted to be benign. The remaining mutations are predicted to cause substantial changes in the protein structure. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature. Conclusions Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi colorectal cancer patients may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients of various ethnicities, particularly beyond the most common hotspot alleles in exons 2 and 3 is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis.

Chapter 25 – Genetics of Paget’s Disease of Bone

Genetic factors play a central role in Paget’s disease of bone (PDB). Mutations of SQSTM1 are the most common cause of familial PDB. Mutations of TNFRSF11A cause familial expansile osteolysis and related disorders, which present during childhood with deafness and adolescence with a PDB-like phenotype. Mutations of TNFRSF11B cause the recessively inherited condition of juvenile PDB. Mutations of VCP, HNRNPA2B1, and HNRNPA1 cause syndromes in which PDB is associated with myopathy and neurodegeneration. Finally, several common genetic variants have been identified at the TNFRSF11A, CSF1, OPTN, DCSTAMP, RIN3, and PML loci, which act additively to influence the occurrence and severity of developing classical PDB. Environmental influences interact with genetic background to influence susceptibility to PDB by mechanisms that are incompletely understood. Advances in PDB genetics have improved understanding of the mechanisms by which bone remodeling is regulated and have uncovered genetic markers of disease occurrence and severity that might be of clinical value.

Novel KRAS gene mutations in Saudi colorectal cancer patients

&NA; We report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi colorectal cancer patients from the Eastern Province. Methods: Genomic DNA was extracted from paraffin-embedded, formalin-fixed cancerous and noncancerous colorectal tissues. A nested PCR approach was implemented. Successful and specific PCR products were then bi-directionally sequenced. The functional impact of the novel mutations on the K-ras protein was assessed using bioinformatics tools and molecular modeling. Results: KRAS gene mutations were detected in the cancer tissue of 24 out of 56 cases studied. Of these, 11 were in exon 4 (19.64%). The 11 cases with exon 4 aberrations harbored 8 different mutations. All of these except two altered the K-ras protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. The molecular modeling data fit with the prediction from Polyphen-2 (polymorphism phenotyping v2) and SIFT (sorting intolerant from tolerant tools), as well as conservation data. One mutation is predicted to be benign and modeling also showed little predicted effect on protein structure. The remaining mutations are predicted to cause substantial changes in the protein structure in line with the predicted damaging effect by polyphen-2 software. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.1 Conclusions: Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi patients from the Eastern Province may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to the paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients, particularly beyond the most common hotspot alleles in exons 2 and 3, is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis. ReferencePalmirotta R, Savonarola A, Formica V, et al. A novel K-ras mutation in colorectal cancer. A case report and literature review. Anticancer Res 2009; 29: 3369–74.

Genome wide association study identifies seven loci that account for 86% of the population attributable risk of Paget's disease of bone

Objectives: Recently, we have identified DLKl/FA1 (delta like 1/fetal antigen1) as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage (Harkness L, et al., Stem Cell Rev and Rep, 2009;5:353). We aimed in this study to investigate the regulatory role of Dlk1/FA1 in chondrogenesis. Materials/Methods: We used the mouse embryonic limb mesenchymal micromass culture as an in vitro system that recapitulates the sequential stages of chondrogenesis. In this culture system, we examined the possible regulation of DLK1/ FA1 expression in response to different signal pathways and their antagonists that involved in the entire program of cartilage development. Real-time PCR, immunostaining and ELISA (for active soluble form of DLK1/FA1) assays were used. Results: Our data showed that DLK1/FA1 started to be expressed at stages of mesenchyme condensation (day 1–3) and peaked up through chondrogenesis (day 3–6) in parallel with the expression of Sox9 and type II collagen, while dramatically down-regulated after day 7 to be abolished completely upon the expression of ColX by hypertrophic chondrocyte at day 10. Interestingly, TGF-β1 (an early signal for condensation/differentiation) treatment delays chondrocyte hypertrophy and inhibits matrix mineralization in parallel with marked down-regulation of Dlk1 expression (by 80%) without affecting the expression of early chondrogenic markers Sox9 and Col2a1. In contrast, blocking of TGF-β1 signal using SB431542 strongly inhibits mesenchyme condensation and chondrogenesis in parallel with marked stimulation ofDlk1 expression (by 4-fold), suggesting an involvement of DLK1/FA1 in mediating the function of TGF-β1 signalling in chondrogenesis. In support of this hypothesis, we found that TGF-β1 enhanced chondrocyte differentiation in Dlk1MEF compared to wild type MEF. Conclusions: In conclusion, our data identified TGF-β1 as an upstream negative regulator of Dlk1 expression and function during the early events of embryonic chondrogenesis. The cross-talk between TGF-β1 and DLK1 showed to promote early chondrogenesis during embryonic endochondral bone formation process. Disclosure of Interest: None declared.