Collaborative cross mice in a genetic association study reveal new candidate genes for bone microarchitecture.

BACKGROUND: The microstructure of trabecular bone is a composite trait governed by a complex interaction of multiple genetic determinants. Identifying these genetic factors should significantly improve our ability to predict of osteoporosis and its associated risks. Genetic mapping using collaborative cross mice (CC), a genetically diverse recombinant inbred mouse reference panel, offers a powerful tool to identify causal loci at a resolution under one mega base-pairs, with a relatively small cohort size. Here, we utilized 31 CC lines (160 mice of both sexes in total) to perform genome-wide haplotype mapping across 77,808 single-nucleotide polymorphisms (SNPs). Haplotype scans were refined by imputation with the catalogue of sequence variation segregating in the CC to suggest potential candidate genes. Trabecular traits were obtained following microtomographic analysis, performed on 10-µm resolution scans of the femoral distal metaphysis. We measured the trabecular bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), and connectivity density (Conn.D). RESULTS: Heritability of these traits ranged from 0.6 to 0.7. In addition there was a significant (P < 0.01) sex effect in all traits except Tb.Th. Our haplotype scans yielded six quantitative trait loci (QTL) at 1 % false discovery rate; BV/TV and Tb.Th produced two proximal loci each, on chromosome 2 and 7, respectively, and Tb.N and Conn.D yielded one locus on chromosomes 8 and 14, respectively. We identified candidate genes with previously-reported functions in bone biology, and implicated unexpected genes whose function in bone biology has yet to be assigned. Based on the literature, among the genes that ranked particularly high in our analyses (P < 10(-6)) and which have a validated causal role in skeletal biology, are Avp, Oxt, B2m (associated with BV/TV), Cnot7 (with Tb.N), Pcsk6, Rgma (with Tb.Th), Rb1, and Cpb2 (with Conn.D). Other candidate genes strongly suggested by our analyses are Sgcz, Fgf20 (associated with Tb.N), and Chd2 (with Tb.Th). CONCLUSION: We have demonstrated for the first time genome-wide significant association between several genetic loci and trabecular microstructural parameters for genes with previously reported experimental observations, as well as proposing a role for new candidate genes with no previously characterized skeletal function.

A study of the influence of genetic variance and sex on the density and thickness of the calvarial bone in collaborative cross mice.

BACKGROUND: Bone microarchitecture is affected by multiple genes, each having a small effect on the external appearance. It is thus challenging to characterize the genes and their specific effect on bone thickness and porosity. The purpose of this study was to assess the heritability and the genetic variation effect, as well as the sex effect on the calvarial bone thickness (Ca.Th) and calvarial porosity (%PoV) using the Collaborative Cross (CC) mouse population. METHODS: In the study we examined the parietal bones of 56 mice from 9 lines of CC mice. Morphometric parameters were evaluated using microcomputed tomography (µCT) and included Ca.Th and %PoV. We then evaluated heritability, genetic versus environmental variance and the sex effect for these parameters. RESULTS: Our morphometric analysis showed that Ca.Th and %PoV are both significantly different among the CC lines with a broad sense heritability of 0.78 and 0.90, respectively. The sex effect within the lines was significant in line IL111 and showed higher values of Ca.Th and %PoV in females compared to males. In line IL19 there was a borderline sex effect in Ca.Th in which males showed higher values than females. CONCLUSIONS: These results stress the complexity of sex and genotype interactions controlling Ca.Th and %PoV, as the skeletal sexual dimorphism was dependent on the genetic background. This study also shows that the CC population is a powerful tool for establishing the genetic effect on these traits.

A genome-wide association study in mice reveals a role for Rhbdf2 in skeletal homeostasis.

Low bone mass and an increased risk of fracture are predictors of osteoporosis. Individuals who share the same bone-mineral density (BMD) vary in their fracture risk, suggesting that microstructural architecture is an important determinant of skeletal strength. Here, we utilized the rich diversity of the Collaborative Cross mice to identify putative causal genes that contribute to the risk of fractures. Using microcomputed tomography, we examined key structural features that pertain to bone quality in the femoral cortical and trabecular compartments of male and female mice. We estimated the broad-sense heritability to be 50-60% for all examined traits, and we identified five quantitative trait loci (QTL) significantly associated with six traits. We refined each QTL by combining information inferred from the ancestry of the mice, ranging from RNA-Seq data and published literature to shortlist candidate genes. We found strong evidence for new candidate genes, particularly Rhbdf2, whose close association with the trabecular bone volume fraction and number was strongly suggested by our analyses. We confirmed our findings with mRNA expression assays of Rhbdf2 in extreme-phenotype mice, and by phenotyping bones of Rhbdf2 knockout mice. Our results indicate that Rhbdf2 plays a decisive role in bone mass accrual and microarchitecture.

Quantitative mass spectrometry analysis reveals a panel of nine proteins as diagnostic markers for colon adenocarcinomas.

Adenocarcinomas are cancers originating from the gland forming cells of the colon and rectal lining, and are known to be the most common type of colorectal cancers. The current diagnosis strategies for colorectal cancers include biopsy, laboratory tests, and colonoscopy which are time consuming. Identification of protein biomarkers could aid in the detection of colon adenocarcinomas (CACs). In this study, tissue proteome of colon adenocarcinomas (n = 11) was compared with the matched control specimens (n = 11) using isobaric tags for relative and absolute quantitation (iTRAQ) based liquid chromatography-mass spectrometry (LC-MS/MS) approach. A list of 285 significantly altered proteins was identified in colon adenocarcinomas as compared to its matched controls, which are associated with growth and malignancy of the tumors. Protein interaction analysis revealed the association of altered proteins in colon adenocarcinomas with various transcription factors and their targets. A panel of nine proteins was validated using multiple reaction monitoring (MRM). Additionally, S100A9 was also validated using immunoblotting. The identified panel of proteins may serve as potential biomarkers and thereby aid in the detection of colon adenocarcinomas.