Identification of Candidate Gene Regions in the Rat by Co-Localization of QTLs for Bone Density, Size, Structure and Strength.

Susceptibility to osteoporotic fracture is influenced by genetic factors that can be dissected by whole-genome linkage analysis in experimental animal crosses. The aim of this study was to characterize quantitative trait loci (QTLs) for biomechanical and two-dimensional dual-energy X-ray absorptiometry (DXA) phenotypes in reciprocal F2 crosses between diabetic GK and normo-glycemic F344 rat strains and to identify possible co-localization with previously reported QTLs for bone size and structure. The biomechanical measurements of rat tibia included ultimate force, stiffness and work to failure while DXA was used to characterize tibial area, bone mineral content (BMC) and areal bone mineral density (aBMD). F2 progeny (108 males, 98 females) were genotyped with 192 genome-wide markers followed by sex- and reciprocal cross-separated whole-genome QTL analyses. Significant QTLs were identified on chromosome 8 (tibial area; logarithm of odds (LOD) = 4.7 and BMC; LOD = 4.1) in males and on chromosome 1 (stiffness; LOD = 5.5) in females. No QTLs showed significant sex-specific interactions. In contrast, significant cross-specific interactions were identified on chromosome 2 (aBMD; LOD = 4.7) and chromosome 6 (BMC; LOD = 4.8) for males carrying F344mtDNA, and on chromosome 15 (ultimate force; LOD = 3.9) for males carrying GKmtDNA, confirming the effect of reciprocal cross on osteoporosis-related phenotypes. By combining identified QTLs for biomechanical-, size- and qualitative phenotypes (pQCT and 3D CT) from the same population, overlapping regions were detected on chromosomes 1, 3, 4, 6, 8 and 10. These are strong candidate regions in the search for genetic risk factors for osteoporosis

Genetics of Osteoporosis - Studies on bone size, structure and strength in the rat

Osteoporosis is characterized by decreased bone mineral density (BMD) leading to reduced bone strength and increased fracture risk. Several heritable components affect a bone’s ability to resist fracture, including size, structure and strength; therefore identification of the genes underlying several bone characteristics will help elucidate the pathogenesis of fracture risk. Dissection of the genetic determinants of osteoporosis has been more effective in animal models than in human populations due to the possibility of environmental control and minimization of genetic heterogeneity. The aim of this thesis was to identify quantitative trait loci (QTLs) affecting osteoporosis-related phenotypes in an F2 intercross between diabetic GK and non-diabetic F344 rats, differing in their mitochondrial (mt) DNA. For bone measurements, tibia were characterized using four different methods generating several skeletal determinants of fracture risk. Comprehensive analysis identified several chromosomal regions linked to bone size, structure and strength that were influenced by both sex- and reciprocal cross. A region on chromosome 1 was identified with linkage to several bone phenotypes and also fasting glucose, making this region as a strong candidate for the localisation of genes contributing to bone regulation and potentially type-2 diabetes. The observed interaction between nuclear QTLs for bone phenotypes and reciprocal cross, demonstrates a new interesting aspect when interpreting the genetics of phenotypes related to bone strength. Furthermore, preliminary studies suggest that this rat model can be a useful tool to delineate the genetics of type-2 diabetes and osteoporosis in conjunction with lifestyle factors such as high-fat diet

Non-invasive detection of c-myc p64, c-myc p67 and c-erbb-2 in colorectal cancer

Objective. Colorectal cancer is a major cause of cancer mortality in the industrialized nations in the West. Because mortality is closely related to the stage of the disease at the time of diagnosis, detection at an early stage is likely to result in improved recovery rates. Since current diagnostic procedures such as colonoscopy are invasive and the fecal occult blood test (FOBT) lacks sensitivity and specificity for the detection of early lesions, the development of non-invasive methods based on molecular markers of neoplasia can lead to earlier diagnosis and more favorable outcomes for patients with colorectal cancer. Recent advances in the technology for isolating colonocytes from stool (SCSR ( somatic cell sampling and recovery)) provide a non-invasive tool for the study of biomarkers expressed in colorectal cancer. The aim of this study was to detect mRNA expression of three biomarkers: (c-erbb-2 and two forms of c-myc: p64 and p67) in fecal colonocytes and to evaluate its use in diagnosing colorectal cancer. Material and methods. Colonocytes ( SCSR cells) were isolated from stools from 30 subjects: 15 colorectal cancer patients and 15 normal controls. One cancer patient was excluded from the final data analysis because the tumor was a gastrointestinal lymphoma. Each sample yielded two fractions: a pellet and an interphase. Expression of c-myc p64, c-myc p67 and c-erbb-2 mRNA was evaluated in each of the fractions by reverse transcriptase-polymerase chain reaction (RT-PCR). A marker was considered positive upon detecting an amplicon of the expected size in agarose gel electrophoresis. Results. c-myc p64 mRNA expression was observed in both fractions in 78.5% of colorectal cancer patients, compared with 13.3% in the control group ( p = 0.009). For c-myc p67, 78.6% of the colorectal cancer patients showed mRNA expression in both fractions in comparison with only 13.3% of the controls ( p = 0.003). C-erbb-2 showed no significant difference in mRNA expression between colorectal cancer and controls. When the data were analyzed for co-expression of c-myc p64 and c-myc p67, in both pellet and interphase, sensitivity was 64% and specificity was 100%. Conclusions. Fecal colonocytes isolated by somatic cell sampling and recovery ( SCSR) technology could be used for the non-invasive assessment of the expression of biomarkers of colon cancer such as c-myc p64, c-myc p67 and c-erbb-2. The expression of c-myc p64 and c-myc p67 in colonocytes showed a significant association with colorectal cancer and may be helpful as a biomarker for the non-invasive detection of colorectal cancer

Genetic loci for bone architecture determined by three-dimensional CT in crosses with the diabetic GK rat.

The F344 rat carries alleles contributing to bone fragility while the GK rat spontaneously develops type-2 diabetes. These characteristics make F344xGK crosses well suited for the identification of genes related to bone size and allow for future investigation on the association with type-2 diabetes. The aim of this study was to identify quantitative trait loci (QTLs) for bone size phenotypes measured by a new application of three-dimensional computed tomography (3DCT) and to investigate the effects of sex- and reciprocal cross. Tibia from male and female GK and F344 rats, representing the parental, F1 and F2 generations, were examined with 3DCT and analyzed for: total and cortical volumetric BMD, straight and curved length, peri- and endosteal area at mid-shaft. F2 progeny (108 male and 98 female) were genotyped with 192 genome-wide microsatellite markers (average distance 10cM). Sex- and reciprocal cross-separated QTL analyses were performed for the identification of QTLs linked to 3DCT phenotypes and true interactions were confirmed by likelihood ratio analysis in all F2 animals. Several genome-wide significant QTLs were found in the sex- and reciprocal cross-separated progeny on chromosomes (chr) 1, 3, 4, 9, 10, 14, and 17. Overlapping QTLs for both males and females in the (GKxF344)F2 progeny were located on chr 1 (39-67cM). This region confirms previously reported pQCT QTLs and overlaps loci for fasting glucose. Sex separated linkage analysis confirmed a male specific QTL on chr 9 (67-82cM) for endosteal area at the fibula site. Analyses separating the F2 population both by sex and reciprocal cross identified cross specific QTLs on chr 14 (males) and chr 3 and 4 (females). Two loci, chr 4 and 6, are unique to 3DCT and separate from pQCT generated loci. The 3DCT method was highly reproducible and provided high precision measurements of bone size in the rat enabling identification of new sex- and cross-specific loci. The QTLs on chr 1 indicate potential genetic association between bone-related phenotypes and traits affecting type-2 diabetes. The results illustrate the complexity of the genetic architecture of bone size phenotypes and demonstrate the importance of complementary methods for bone analysis

Genetic Regulation of Bone Traits Is Influenced by Sex and Reciprocal Cross in F-2 Progeny From GK and F344 Rats

A genome-wide linkage analysis to identify quantitative trait loci (QTLs) for bone phenotypes was performed in an F-2 intercross of inbred spontaneously type 2 diabetic GK and normoglycemic F344 rats (108 males and 98 females). The aim of the study was to locate genome regions with candidate genes affecting trabecular and cortical bone and to investigate the effects of sex and reciprocal cross. pQCT was used to determine tibia] bone phenotypes in the F2 rats, comprising reciprocal crosses with divergent mitochondrial (mt) DNA. Sex and reciprocal cross-separated QTL analyses were performed followed by assessment of specific interactions. Four genome-wide significant QTLs linked to either cortical vBMD, tibia length, body length, or metaphyseal area were identified in males on chromosomes (chr) 1, 8, and 15. In females, three significant QTLs linked to cortical BMC or metaphyseal total vBMD were identified on chr 1 and 2. Several additional suggestive loci for trabecular and cortical traits were detected in both males and females. Four female-specific QTLs on chr 2, 3, 5, and 10 and four reciprocal cross-specific QTLs on chr 1, 10, and 18 were identified, suggesting that both sex and mt genotype influence the expression of bone phenotypes. J Bone Miner Res 2009;24:1066-1074. Published online on December 29, 2008; doi: 10.1359/JBMR.08125