Predicting candidate genes for human deafness disorders: a bioinformatics approach

BACKGROUND: There are more than 50 genes for autosomal dominant and autosomal recessive nonsyndromic hereditary deafness that are yet to be cloned. The human genome sequence and expression profiles of transcripts in the inner ear have aided positional cloning approaches. The knowledge of protein interactions offers additional advantages in selecting candidate genes within a mapped region. RESULTS: We have employed a bioinformatic approach to assemble the genes encoded by genomic regions that harbor various deafness loci. The genes were then in silico analyzed for their candidacy by expression pattern and ability to interact with other proteins. Such analyses have narrowed a list of 2400 genes from suspected regions of the genome to a manageable number of about 140 for further analysis. CONCLUSION: We have established a list of strong candidate genes encoded by the regions linked to various nonsyndromic hereditary hearing loss phenotypes by using a novel bioinformatic approach. The candidates presented here provide a starting point for mutational analysis in well-characterized families along with genetic linkage to refine the loci. The advantages and shortcomings of this bioinformatic approach are discussed

Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy. Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups. Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin. Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs. Diabetes has emerged as a major worldwide public health concern, and the number of diabetics is estimated to exceed 400 million by the year 203

EphB6 Receptor Modulates Micro RNA Profile of Breast Carcinoma Cells

Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways

Genetic components in diabetic retinopathy

Diabetic retinopathy (DR) is a serious complication of diabetes, which is fast reaching epidemic proportions worldwide. While tight glycemic control remains the standard of care for preventing the progression of DR, better insights into DR etiology require understanding its genetic basis, which in turn may assist in the design of novel treatments. During the last decade, genomic medicine is increasingly being applied to common multifactorial diseases such as diabetes and age-related macular degeneration. The contribution of genetics to the initiation and progression of DR has been recognized for some time, but the involvement of specific genes and genetic variants remains elusive. Several investigations are currently underway for identifying DR susceptibility loci through linkage studies, candidate gene approaches, and genome-wide association studies. Advent of next generation sequencing and high throughput genomic technologies, development of novel bioinformatics tools and collaborations among research teams should facilitate such investigations. Here, we review the current state of genetic studies in DR and discuss reported findings in the context of biochemical, cell biological and therapeutic advances. We propose the development of a consortium in India for genetic studies with large cohorts of patients and controls from limited geographical areas to stratify the impact of the environment. Uniform guidelines should be established for clinical phenotyping and data collection. These studies would permit identification of genetic loci for DR susceptibility in the Indian population and should be valuable for better diagnosis and prognosis, and for clinical management of this blinding disease

Differential Expression Patterns of Eph Receptors and Ephrin Ligands in Human Cancers

Eph receptors constitute the largest family of receptor tyrosine kinases, which are activated by ephrin ligands that either are anchored to the membrane or contain a transmembrane domain. These molecules play important roles in the development of multicellular organisms, and the physiological functions of these receptor-ligand pairs have been extensively documented in axon guidance, neuronal development, vascular patterning, and inflammation during tissue injury. The recognition that aberrant regulation and expression of these molecules lead to alterations in proliferative, migratory, and invasive potential of a variety of human cancers has made them potential targets for cancer therapeutics. We present here the involvement of Eph receptors and ephrin ligands in lung carcinoma, breast carcinoma, prostate carcinoma, colorectal carcinoma, glioblastoma, and medulloblastoma. The aberrations in their abundances are described in the context of multiple signaling pathways, and differential expression is suggested as the mechanism underlying tumorigenesis

Heatmap of altered miRNAs in EphB6-transfected MDA-MB-231 cells.

<p>A two-way hierarchical clustering was performed to generate the heatmap. Each row represents a miRNA and each column represents a sample, and the miRNA clustering tree is shown on the left. Red color represents an expression level above mean and blue color represents expression level lower than the mean. Top 50 miRNAs altered, based on variation across replicates, with a p-value of <0.001 for three vector-transfectants and three EphB6 transfectants are shown.</p

Coordinated changes in the levels of members of specific miRNA families.

<p>The levels of expression for two miRNA clusters in three vector-transfectants and three EphB6-transfectants are shown.</p

Altered miRNAs in EphB6-transfected MDA-MB-231 cells.

@<p>Array hybridizations were performed with RNA isolated from three independent clones of vector-transfected and EphB6-transfected DNA as described in the Methods' section. The altered RNA at a p-value of <0.001 are presented here.</p

Amplification of mRNAs targeted by various miRNAs.

<p>Each panel shows a representative gel picture for the transcript levels in empty vector-transfected (left lane) and EphB6-transfected (right lane) MDA-MB-231 cells. The bar diagram on the right shows quantification of three gels for the levels of transcripts. The Y-axis represents arbitrary units of intensity. Empty vector-transfected and EphB6-transfected MDA-MB-231 cells are designated as −Eph and +Eph. The transcript designations are indicated in each panel. The graph shows linear and comparable amplification of actin transcript in RNA isolated from empty vector-transfected and EphB6-transfected MDA-MB-231 cells.</p

Sequences of primers used for PCR.

<p>Sequences of primers used for PCR.</p