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Acyl-CoA dehydrogenase 9 (ACAD9), linked to chromosome 3q21.3, is one of a family of multimeric mitochondrial flavoenzymes that catalyze the degradation of fatty acyl-CoA from the carnitine shuttle via β-oxidation (He et al. 2007). ACAD9, specifically, is implicated in the processing of palmitoyl-CoA and long-chain unsaturated substrates, but unlike other acyl-CoA dehydrogenases (ACADs), it has a significant role in mitochondrial complex I assembly (Nouws et al. 2010 & 2014). Mutations in this enzyme typically cause mitochondrial complex I deficiency, as well as a mild defect in long chain fatty acid metabolism (Haack et al. 2010, Kirby et al. 2004, Mcfarland et al. 2003, Nouws et al. 2010 & 2014). The clinical phenotype of ACAD9 deficiency and the associated mitochondrial complex I deficiency reflect this unique duality, and symptoms are variable in severity and onset. Patients classically present with cardiac dysfunction due to hypertrophic cardiomyopathy. Other common features include Leigh syndrome, macrocephaly, and liver disease (Robinson et al. 1998). We report the case of an 11-month old girl presenting with microcephaly, dystonia, and lactic acidosis, concerning for a mitochondrial disorder, but atypical for ACAD9 deficiency. Muscle biopsy showed mitochondrial proliferation, but normal mitochondrial complex I activity. The diagnosis of ACAD9 deficiency was not initially considered, due both to these findings and to her atypical presentation. Biochemical assay for ACAD9 deficiency is not clinically available. Family trio-based whole exome sequencing (WES) identified 2 compound heterozygous mutations in the ACAD9 gene. This discovery led to optimized treatment of her mitochondrial dysfunction, and supplementation with riboflavin, resulting in clinical improvement. There have been fewer than 25 reported cases of ACAD9 deficiency in the literature to date. We review these and compare them to the unique features of our patient. ACAD9 deficiency should be considered in the differential diagnosis of patients with lactic acidosis, seizures, and other symptoms of mitochondrial disease, including those with normal mitochondrial enzyme activities. This case demonstrates the utility of WES, in conjunction with biochemical testing, for the appropriate diagnosis and treatment of disorders of energy metabolism.

The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. We have now developed a new ‘BACarray’ methodology, based on affinity purification of polysomal mRNAs from genetically defined cell populations. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. Even two morphologically indistinguishable subclasses of MSNs display vastly different translational profiles. Striatopallidal neurons are characterized by a strong and cell-specific release of intracellular Ca2+ in response to sphingosine 1-phosphate, consistent with their selective expression of Gpr6. In contrast, striatonigral neurons demonstrate a selective cell-specific increase in GABAA receptor subunits in response to chronic cocaine treatment. BACarray translational profiling is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.

Certain hexavalent chromium [Cr(VI)] compounds are known genotoxic respiratory carcinogens, which induce apoptosis as a predominant mode of cell death. Selection of cells that are resistant to apoptosis may be a factor in tumour progression. We developed sub-populations of telomerase-transfected human fibroblasts (BJ-hTERT) that survived a 99% clonogenically lethal exposure to Cr(VI) (B-5Cr). B-5Cr cells were markedly resistant to apoptosis induced by several agents and exhibited increased clonogenic survival, especially at apoptogenic doses. B-5Cr cells did not exhibit altered cellular uptake of Cr(VI) and retained a normal p53 response to Cr(VI) exposure. We conducted large-scale gene expression analysis at different time-points after a secondary genotoxic Cr(VI) insult in B-5Cr and BJ-hTERT cells using Affymetrix Genechip® human genome arrays. Cr(VI) exposure led to differential regulation of many genes, which affect a diverse set of cellular activities such as transcription, signal transduction, stress response, cell adhesion, DNA repair, apoptosis and cell cycle modulation. We compared Cr(VI)-induced altered gene expression in the B-5Cr cells to that in the parental cells and identified 223, 147 and 204 genes with at least a two-fold difference in expression at 4, 8 and 18 h after exposure, respectively. Cluster analysis by gene function revealed altered expression of genes involved in apoptosis, cell cycle regulation and DNA repair. Our data suggest an alteration in gene expression that may favor cell survival and/or incomplete DNA repair after genotoxic exposure. Selection of cells with altered expression of these genes may constitute the early stages of tumour progression.

Purpose To determine the genomic and proteomic expression changes in human trabecular meshwork cells when they are treated with transforming growth factor (TGF)-beta. Methods Human trabecular meshwork cells from five donors were cultured for 3 days with 1 ng/mL of either TGF-beta1 or -beta2. Changes in gene expression determined with gene microarrays and alterations in protein expression detected by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) were studied in these cells after the incubation. Results With both TGF-betas, there was a substantial upregulation of genes that were related to secreted proteins or extracellular matrix. This result was consistent with pathologic changes observed in disease and with experiments on perfused trabecular meshwork. Several of the gene changes suggest that other signaling pathways, such as ErbB and Wnt, were altered. Changes in enzyme expression in the prostaglandin pathway indicated that the prostaglandins may have a different cellular profile in the presence of glaucoma. Two genes, osteoblast-specific factor 2 and corneal-derived transcript 6, which are highly expressed in the cells under normal conditions, were substantially upregulated with the TGF-betas. Proteomic analysis indicated that there was increased proteolysis of vimentin with both treatments. Tropomyosin 1alpha was increased in both gene and protein expression, suggesting alterations of the cytoskeleton by the disease. The TGF-beta1 treatment caused more robust changes than those induced by TGF-beta2. Three genes-aldose reductase, thioredoxin reductase 1, and glucose-6-phosphate 1-dehydrogenase-were identified that were downregulated in expression. These genes had decreases in protein expression with TGF-beta1 treatment but had little change in either gene or protein expression with TGF-beta2. Conclusions Human trabecular meshwork cells can be subjected to increased levels of TGF-beta for several years as a result of glaucoma. The results indicate that changes in extracellular matrix as well as alterations in cytoskeletal proteins occur in these cells as a result of increased TGF-beta. These results are consistent with changes observed in the trabecular meshwork in glaucoma and suggest that at least some of the histologic alterations observed in the meshwork in glaucoma may be the result of increased TGF-betas.

This article identifies the underlying molecular events responsible for fracture nonunions in a subset of fracture patients. Expression profiling of fracture callus tissue from both uneventful fracture repair and nonunion outcomes showed a decrease of COX-2 expression and an inability to mount an immune response in nonunion fractures. Validation in vitro with Saos-2 osteoprogenitor cell lines showed a decrease in osteogenesis potential after the cells were treated with celecoxib, a COX-2 specific inhibitor and anti-inflammatory agent. This article recapitulates that an initial immune response is crucial to fracture healing and suggests limited usage of COX-2 inhibitors in patients with healing fractures.

A causal role of mutations in multiple general transcription factors in neurodevelopmental disorders including autism suggested that alterations in global levels of gene expression regulation might also relate to disease risk in sporadic cases of autism. This premise can be tested by evaluating for changes in the overall distribution of gene expression levels. For instance, in mice, variability in hippocampal-dependent behaviors was associated with variability in the pattern of the overall distribution of gene expression levels, as assessed by variance in the distribution of gene expression levels in the hippocampus. We hypothesized that a similar change in variance might be found in children with autism. Gene expression microarrays covering greater than 47,000 unique RNA transcripts were done on RNA from peripheral blood lymphocytes (PBL) of children with autism (n = 82) and controls (n = 64). Variance in the distribution of gene expression levels from each microarray was compared between groups of children. Also tested was whether a risk factor for autism, increased paternal age, was associated with variance. A decrease in the variance in the distribution of gene expression levels in PBL was associated with the diagnosis of autism and a risk factor for autism, increased paternal age. Traditional approaches to microarray analysis of gene expression suggested a possible mechanism for decreased variance in gene expression. Gene expression pathways involved in transcriptional regulation were down-regulated in the blood of children with autism and children of older fathers. Thus, results from global and gene specific approaches to studying microarray data were complimentary and supported the hypothesis that alterations at the global level of gene expression regulation are related to autism and increased paternal age. Global regulation of transcription, thus, represents a possible point of convergence for multiple etiologies of autism and other neurodevelopmental disorders.

Fracture healing requires controlled expression of thousands of genes. Only a small fraction of these genes have been isolated and fewer yet have been shown to play a direct role in fracture healing. The purpose of this study was threefold: (1) to develop a reproducible open femur model of fracture healing that produces consistent fracture calluses for subsequent RNA extraction, (2) to use this model to determine temporal expression patterns of known and unknown genes using DNA microarray expression profiling, and (3) to identify and validate novel gene expression in fracture healing. In the initial arm of the study, a total of 56 wild-type C57BL/6 mice were used. An open, stabilized diaphyseal femur fracture was created. Animals were killed at 1, 5, 7, 10, 14, 21, and 35 days after surgery and the femurs were harvested for analysis. At each time point, fractures were radiographed and sectioned for histologic analyses. Tissue from fracture callus at all stages following fracture yielded reproducibly large amounts of mRNA. Expression profiling revealed that genes cluster by function in a manner similar to the histologic stages of fracture healing. Based on the expression profiling of fracture tissue, temporal expression patterns of several genes known to be involved in fracture healing were verified. Novel expression of multiple genes in fracture callous tissue was also revealed including leptin and leptin receptor. In order to test whether leptin signaling is required for fracture repair, mice deficient in leptin or its receptor were fractured using the same model. Fracture calluses of mice deficient in both leptin or leptin receptor are larger than wild-type mice fractures, likely due to a delay in mineralization, revealing a previously unrecognized role of leptin signaling in fracture healing. This novel model of murine fracture repair is useful in examining both global changes in gene expression as well as individual signaling pathways, which can be used to identify specific molecular mechanisms of fracture healing.

Pterygium is a sunlight-related, ocular-surface lesion that can obscure vision. In order to identify specific genes that may play a role in pterygium pathogenesis, we analyzed the global gene expression profile of pterygium in relation to autologous conjunctiva.Oligonucleotide microarray hybridization was used to compare the gene expression profile between human whole pterygium and autologous conjunctiva. Selected genes were further characterized by RT-PCR, western blot, and immunohistochemistry, and comparisons were made with limbal and corneal tissues.Thirty-four genes exhibited a 2 fold or greater difference in expression between human whole pterygium and autologous conjunctiva. Twenty-nine transcripts were increased and five transcripts were decreased in pterygium. Fibronectin, macrophage-inflammatory protein-4 (MIP-4), and lipocalin 2 (oncogene 24p3; NGAL) were increased 9, 5, and 2.4 fold, respectively, while Per1 and Ephrin-A1 were decreased 2 fold in pterygium. Western blots showed that fibronectin and MIP-4 were increased in pterygium compared to limbus, cornea, and conjunctiva. Immunohistochemical analysis showed fibronectin in the stroma; lipocalin 2 in the basal epithelial cells, basement membrane, and extracellular stroma; and MIP-4 in all areas of the pterygium.These data show both novel and previously identified extracellular-matrix-related, proinflammatory, angiogenic, fibrogenic, and oncogenic genes expressed in human pterygium. Comparisons of selected genes with limbal and corneal tissues gave results similar to comparisons between pterygium and normal conjunctiva. The increased expression of lipocalin 2, which activates matrix metalloproteinases (MMP), is consistent with our previous findings that MMP-9 and other MMPs are highly expressed in pterygium basal epithelium.

Neurofibrillary tangles (NFT) constitute one of the cardinal histopathological features of Alzheimer's disease (AD). To explore in vivo molecular processes involved in the development of NFTs, we compared gene expression profiles of NFT-bearing entorhinal cortex neurons from 19 AD patients, adjacent non-NFT-bearing entorhinal cortex neurons from the same patients, and non-NFT-bearing entorhinal cortex neurons from 14 non-demented, histopathologically normal controls (ND). Of the differentially expressed genes, 225 showed progressively increased expression (AD NFT neurons > AD non-NFT neurons > ND non-NFT neurons) or progressively decreased expression (AD NFT neurons < AD non-NFT neurons < ND non-NFT neurons), raising the possibility that they may be related to the early stages of NFT formation. Immunohistochemical studies confirmed that many of the implicated proteins are dysregulated and preferentially localized to NFTs, including apolipoprotein J, interleukin-1 receptor-associated kinase 1, tissue inhibitor of metalloproteinase 3, and casein kinase 2, beta. Functional validation studies are underway to determine which candidate genes may be causally related to NFT neuropathology, thus providing therapeutic targets for the treatment of AD.

Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFβ1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor–driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.