Cartilage-selective genes identified in genome-scale analysis of non-cartilage and cartilage gene expression

<p>Abstract</p> <p>Background</p> <p>Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate into chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18–22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed Celsius.</p> <p>Results</p> <p>161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as cartilage collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis.</p> <p>Conclusion</p> <p>Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated.</p

Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing

<p>Abstract</p> <p>Background</p> <p>The emergence of next-generation sequencing technology presents tremendous opportunities to accelerate the discovery of rare variants or mutations that underlie human genetic disorders. Although the complete sequencing of the affected individuals' genomes would be the most powerful approach to finding such variants, the cost of such efforts make it impractical for routine use in disease gene research. In cases where candidate genes or loci can be defined by linkage, association, or phenotypic studies, the practical sequencing target can be made much smaller than the whole genome, and it becomes critical to have capture methods that can be used to purify the desired portion of the genome for shotgun short-read sequencing without biasing allelic representation or coverage. One major approach is array-based capture which relies on the ability to create a custom in-situ synthesized oligonucleotide microarray for use as a collection of hybridization capture probes. This approach is being used by our group and others routinely and we are continuing to improve its performance.</p> <p>Results</p> <p>Here, we provide a complete protocol optimized for large aggregate sequence intervals and demonstrate its utility with the capture of all predicted amino acid coding sequence from 3,038 human genes using 241,700 60-mer oligonucleotides. Further, we demonstrate two techniques by which the efficiency of the capture can be increased: by introducing a step to block cross hybridization mediated by common adapter sequences used in sequencing library construction, and by repeating the hybridization capture step. These improvements can boost the targeting efficiency to the point where over 85% of the mapped sequence reads fall within 100 bases of the targeted regions.</p> <p>Conclusions</p> <p>The complete protocol introduced in this paper enables researchers to perform practical capture experiments, and includes two novel methods for increasing the targeting efficiency. Coupled with the new massively parallel sequencing technologies, this provides a powerful approach to identifying disease-causing genetic variants that can be localized within the genome by traditional methods.</p

Ciliary Abnormalities Due to Defects in the Retrograde Transport Protein DYNC2H1 in Short-Rib Polydactyly Syndrome

The short-rib polydactyly (SRP) syndromes are a heterogenous group of perinatal lethal skeletal disorders with polydactyly and multisystem organ abnormalities. Homozygosity by descent mapping in a consanguineous SRP family identified a genomic region that contained DYNC2H1, a cytoplasmic dynein involved in retrograde transport in the cilium. Affected individuals in the family were homozygous for an exon 12 missense mutation that predicted the amino acid substitution R587C. Compound heterozygosity for one missense and one null mutation was identified in two additional nonconsanguineous SRP families. Cultured chondrocytes from affected individuals showed morphologically abnormal, shortened cilia. In addition, the chondrocytes showed abnormal cytoskeletal microtubule architecture, implicating an altered microtubule network as part of the disease process. These findings establish SRP as a cilia disorder and demonstrate that DYNC2H1 is essential for skeletogenesis and growth

Disease Gene Characterization through Large-Scale Co-Expression Analysis

In the post genome era, a major goal of biology is the identification of specific roles for individual genes. We report a new genomic tool for gene characterization, the UCLA Gene Expression Tool (UGET).Celsius, the largest co-normalized microarray dataset of Affymetrix based gene expression, was used to calculate the correlation between all possible gene pairs on all platforms, and generate stored indexes in a web searchable format. The size of Celsius makes UGET a powerful gene characterization tool. Using a small seed list of known cartilage-selective genes, UGET extended the list of known genes by identifying 32 new highly cartilage-selective genes. Of these, 7 of 10 tested were validated by qPCR including the novel cartilage-specific genes SDK2 and FLJ41170. In addition, we retrospectively tested UGET and other gene expression based prioritization tools to identify disease-causing genes within known linkage intervals. We first demonstrated this utility with UGET using genetically heterogeneous disorders such as Joubert syndrome, microcephaly, neuropsychiatric disorders and type 2 limb girdle muscular dystrophy (LGMD2) and then compared UGET to other gene expression based prioritization programs which use small but discrete and well annotated datasets. Finally, we observed a significantly higher gene correlation shared between genes in disease networks associated with similar complex or Mendelian disorders.UGET is an invaluable resource for a geneticist that permits the rapid inclusion of expression criteria from one to hundreds of genes in genomic intervals linked to disease. By using thousands of arrays UGET annotates and prioritizes genes better than other tools especially with rare tissue disorders or complex multi-tissue biological processes. This information can be critical in prioritization of candidate genes for sequence analysis

Comprehensive assessment of expression of insulin signaling pathway components in subcutaneous adipose tissue of women with and without polycystic ovary syndrome

Objective: Insulin resistance is a common feature of polycystic ovary syndrome (PCOS). The insulin signaling pathway consists of two major pathways, the metabolic and the mitogenic cascades. The many components of these pathways have not been comprehensively analyzed for differential expression in insulin-responsive tissues in PCOS. The goal of this study was to determine whether the core elements of the insulin signal transduction cascade were differentially expressed in subcutaneous adipose tissue (SAT) between PCOS and controls. Materials/methods: Quantitative real-time PCR for 36 insulin signaling pathway genes was performed in subcutaneous adipose tissue from 22 white PCOS and 13 healthy controls. Results: Genes in the insulin signaling pathway were not differentially expressed in subcutaneous adipose tissue between PCOS and controls (P > 0.05 for all). Components mainly of the mitogenic pathway were correlated with both androgens and metabolic phenotypes. Expression levels of five genes (MKNK1, HRAS, NRAS, KRAS, and GSK3A) were positively correlated with total testosterone level (ρ > 0, P < 0.05). Inverse correlation was found between expression of six genes (HRAS, MAP2K2, NRAS, MAPK3, GRB2, and SHC1) and metabolic traits (body mass index, fasting glucose, fasting insulin, and HOMA-IR) (ρ < 0, P < 0.05). Conclusions: Differential expression of core insulin signaling pathway components in subcutaneous adipose tissue is not a major contributor to the pathogenesis of PCOS. Correlation between clinical phenotypes and expression of several genes in the mitogenic limb of the insulin signaling pathway suggests mitogenic signaling by insulin may regulate steroidogenesis and glucose homeostasis

Differentially Expressed Wound Healing-Related microRNAs in the Human Diabetic Cornea

<div><p>MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) <i>in vitro</i>. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by <i>in situ</i> hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR^TMmiRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.</p> </div

Effect of miRNAs on wound healing.

<p>A. HCEC were transfected with pre-miR-146a, -424, and -145 (top panel) or their antagomirs, pre-miRNA inhibitors (bottom panel); for control, irrelevant Cy3-labeled pre-miRNA was used. HCEC were scratch wounded as indicated in Methods. Wound closure was quantified using ImageJ software at 20 hr after wounding for pre-miRNA transfected cells and at 24 hr for pre-miRNA antagomir transfected cells. Bars represent SEM. *, p < 0.05 vs. miR-Cy3. B. miR-146a decreased wound closure, whereas its antagomir significantly increased it at 20 h compared to control. Values are expressed as percentage of the initial wound area. Data are means of three independent experiments in triplicate. </p