Showing papers by "Noam Shomron published in 2013"

Desmoglein 1 deficiency results in severe dermatitis, multiple allergies and metabolic wasting

Abstract: The relative contribution of immunological dysregulation and impaired epithelial barrier function to allergic diseases is still a matter of debate. Here we describe a new syndrome featuring severe dermatitis, multiple allergies and metabolic wasting (SAM syndrome) caused by homozygous mutations in DSG1. DSG1 encodes desmoglein 1, a major constituent of desmosomes, which connect the cell surface to the keratin cytoskeleton and have a crucial role in maintaining epidermal integrity and barrier function. Mutations causing SAM syndrome resulted in lack of membrane expression of DSG1, leading to loss of cell-cell adhesion. In addition, DSG1 deficiency was associated with increased expression of a number of genes encoding allergy-related cytokines. Our deciphering of the pathogenesis of SAM syndrome substantiates the notion that allergy may result from a primary structural epidermal defect.

Pax6 regulates gene expression in the vertebrate lens through miR-204.

Abstract: During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.

Analysis of insertion–deletion from deep-sequencing data: software evaluation for optimal detection

Abstract: Insertion and deletion (indel) mutations, the most common type of structural variance in the human genome, affect a multitude of human traits and diseases. New sequencing technologies, such as deep sequencing, allow massive throughput of sequence data and greatly contribute to the field of disease causing mutation detection, in general, and indel detection, specifically. In order to infer indel presence (indel calling), the deep-sequencing data have to undergo comprehensive computational analysis. Selecting which indel calling software to use can often skew the results and inherent tool limitations may affect downstream analysis. In order to better understand these inter-software differences, we evaluated the performance of several indel calling software for short indel (1^10 nt) detection. We compared the software’s sensitivity and predictive values in the presence of varying parameters such as read depth (coverage), read length, indel size and frequency. We pinpoint several key features that assist successful experimental design and appropriate tool selection. Our study may also serve as a basis for future evaluation of additional indel calling methods.

microRNA-125a-3p reduces cell proliferation and migration by targeting Fyn

Abstract: Fyn, a member of the Src family kinases (SFKs), has a pivotal role in cell adhesion, proliferation, migration and survival, and its overexpression is associated with several types of cancer. MicroRNAs (miRNAs) play a major role in post-transcriptional repression of protein expression. In light of the significant functions of Fyn, together with studies demonstrating miR-125a as a tumor-suppressing miRNA that is downregulated in several cancer cell types and on our bioinformatics studies presented here, we chose to examine the post-transcription regulation of Fyn by miR-125a-3p in the HEK 293T cell line. We show that Fyn expression can be dramatically reduced by elevated levels of miR-125a-3p. Following this reduction, the activity of proteins downstream of Fyn, such as FAK, paxillin and Akt (proteins known to be overexpressed in various tumors), is also reduced. On a broader level, we show that miR-125a-3p causes an arrest of the cell cycle at the G2/M stage and decreases cell viability and migration, probably in a Fyn-directed manner. The results are reinforced by control experiments conducted using Fyn siRNA and anti-miR-125a-3p, as well as by the fact that numerous cancer cell lines show a significant downregulation of Fyn after mir-125a-3p overexpression. Collectively, we conclude that miR-125a-3p has an important role in the regulation of Fyn expression and of its signaling pathway, which implies that it has a therapeutic potential in overexpressed Fyn-related diseases.

Opposing actions of environmental enrichment and Alzheimer’s disease on the expression of hippocampal microRNAs in mouse models

Abstract: Alzheimer's disease (AD) is the most common form of dementia in the elderly. Although there are no drugs that modify the disease process, exposure to an enriched environment (EE) can slow the disease progression. Here, we characterize the effects of AD and EE on the post-transcriptional regulators, microRNAs (miRNAs), which may contribute to the detrimental and beneficial effects of AD and EE, respectively, on synaptic plasticity-related proteins and AD pathology. We found for the first time miRNAs that were inversely regulated in AD and EE, and may affect synaptic proteins and modulators, molecular factors associated with AD pathology, and survival and neuroprotective factors. MiRNAs that were upregulated only in 3xTgAD mice model of AD compared with their control mice were localized to synapses, predicted to downregulate essential synaptic proteins and are highly associated with regulating apoptosis, AD-associated processes and axon guidance. Studying the progressive change in miRNAs modulation during aging of 3xTgAD mice, we identified miRNAs that were regulated in earlier stages of AD, suggesting them as potential AD biomarkers. Last, we characterized AD- and EE-related effects in the mouse hippocampus on tomosyn protein levels, an inhibitor of the synaptic transmission machinery. While EE reduced tomosyn levels, tomosyn levels were increased in old 3xTgAD mice, suggesting a role for tomosyn in the impairment of synaptic transmission in AD. Interestingly, we found that miR-325 regulates the expression levels of tomosyn as demonstrated by a luciferase reporter assay, and that miR-325 was downregulated in AD and upregulated following EE. These findings improve our understanding of the molecular and cellular processes in AD pathology, following EE, and the interplay between the two processes, and open new avenues for the studies of understanding and controlling AD.

Genome-wide expression profiling of human lymphoblastoid cell lines implicates integrin beta-3 in the mode of action of antidepressants.

Abstract: Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for major depression. However, the link between inhibition of serotonin reuptake and remission from depression remains controversial: in spite of the rapid onset of serotonin reuptake inhibition, remission from depression takes several weeks, presumably reflecting synaptogenesis/neurogenesis and neuronal rewiring. We compared genome-wide expression profiles of human lymphoblastoid cell lines from unrelated individuals following treatment with 1 μM paroxetine for 21 days with untreated control cells and examined which genes and microRNAs (miRNAs) showed the most profound and consistent expression changes. ITGB3, coding for integrin beta-3, showed the most consistent altered expression (1.92-fold increase, P=7.5 × 10−8) following chronic paroxetine exposure. Using genome-wide miRNA arrays, we observed a corresponding decrease in the expression of two miRNAs, miR-221 and miR-222, both predicted to target ITGB3. ITGB3 is crucial for the activity of the serotonin transporter (SERT), the drug target of SSRIs. Moreover, it is presumably required for the neuronal guidance activity of CHL1, whose expression was formerly identified as a tentative SSRI response biomarker. Further genes whose expression was significantly modulated by chronic paroxetine are also implicated in neurogenesis. Surprisingly, the expression of SERT or serotonin receptors was not modified. Our findings implicate ITGB3 in the mode of action of SSRI antidepressants and provide a novel link between CHL1 and the SERT. Our observations suggest that SSRIs may relieve depression primarily by promoting neuronal synaptogenesis/neurogenesis rather than by modulating serotonin neurotransmission per se.

Cole Disease Results from Mutations in ENPP1

Abstract: The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Abstract: Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

Species-specific microRNA regulation influences phenotypic variability: perspectives on species-specific microRNA regulation.

Abstract: Phenotypic divergence among animal species may be due in part to species-specific (SS) regulation of gene expression by small, non-coding regulatory RNAs termed "microRNAs". This phenomenon can be modulated by several variables. First, microRNA genes vary by their level of conservation, many of them being SS, or unique to a particular evolutionary lineage. Second, microRNA expression levels vary spatially and temporally in different species. Lastly, while microRNAs bind the 3'UTR of target genes in order to silence their expression, the binding sites themselves are often non-conserved. The variability of the miRNA-target paradigm between different species is thus multifactorial, and this paradigm has only just started to gain attention from researchers in various fields. Here we present and discuss recent findings regarding the characteristics and implications of SS microRNA regulation.

Involvement of IGF-1R regulation by miR-515-5p modifies breast cancer risk among BRCA1 carriers.

Abstract: Several lines of evidence indicate that sequence alterations within microRNA (miRNA)-binding sites can modify the binding to its target gene resulting in altered expression patterns We hypothesized that a single nucleotide polymorphism (SNP) located in the miR-515-5p binding site of igf-1r gene may alter IGF-1R regulation, with consequent effects on breast cancer risk in BRCA1 mutation carriers Computational prediction revealed that the rs28674628 SNP in the igf-1r 3′ UTR is located within a predicted binding site for miR-515-5p The effect of this SNP on breast cancer risk was evaluated by genotyping 115 Jewish Ashkenazi carriers of the 185delAG mutation in the BRCA1 gene using the Sequenom platform followed by Kaplan–Meier analysis Additional data set of 378 Jewish BRCA1 carriers was analyzed to validate our results MiRNA transfection, Western blot analysis, luciferase reporter assay, real time PCR, and immunohistochemistry were performed to assess direct regulation of igf-1r by miR-515-5p We show direct regulation of IGF-1R by miR-515-5p We identified that disrupting miR-515-5p and igf-1r 3′ UTR binding by SNP may cause elevated IGF-1R protein levels Interestingly, miR-515-5p is downregulated in tumor tissue compared to its non-neoplastic surrounding tissue while IGF-1R levels are elevated This igf-1r SNP was found to be significantly associated with age at diagnosis of breast cancer in Jewish Ashkenazi BRCA1 mutation carriers These findings support the hypothesis that a SNP located in igf-1r gene may alter miRNA regulation of IGF-1R, with a putative effect on BRCA1 penetrance and breast cancer risk

Exome sequencing analysis: a guide to disease variant detection.

Abstract: Whole exome sequencing presents a powerful tool to study rare genetic disorders. The most challenging part of using exome sequencing for the purpose of disease-causing variant detection is analyzing, interpreting, and filtering the large number of detected variants. In this chapter we provide a comprehensive description of the various steps required for such an analysis. We address strategies in selecting samples to sequence, and technical considerations involved in exome sequencing. We then discuss how to identify variants, and methods for first annotating detected variants using characteristics such as allele frequency, location in the genome, and predicted severity, and then classifying and prioritizing the detected variants based on those annotations. Finally, we review possible gene annotations that may help to establish a relationship between genes carrying high-priority variants and the phenotype in question, in order to identify the most likely causative mutations.

Missense mutation in the MEN1 gene discovered through whole exome sequencing co-segregates with familial hyperparathyroidism

Abstract: Familial isolated hyperparathyroidism (FIHP) can be encountered in the context of multiple endocrine neoplasia type 1 (MEN1), hyperparathyroidism and jaw tumour syndrome (HPT-JT) and in familial hypocalciuric hypercalcaemia (FHH). In these syndromes, germline mutations in the relevant genes (MEN1, HPRT2 and CaSR, respectively) are detected. In some FIHP cases, the causative gene is still elusive. The objective of this study is to define the genetic basis of FIHP in a Georgian Jewish family with FIHP using whole exome capture and sequencing. DNA extracted from two sibs and one offspring from a single family all affected with multiglandular hyperparathyroidism was subjected to whole exome capturing and sequencing using the Roche NimbleGen V2 chip and the Illumina HiSeq2000 sequencing platform. Genetic variants were detected and annotated using a combination of the Genome Analysis Tool Kit and in-house scripts. Subsequent confirmation of the mutations and co-segregation analyses were carried out by Sanger sequencing in additional affected and unaffected family members. Whole exome capture and sequencing revealed the collection of variations common to the three-sequenced patients, including a very rare previously described missense mutation (c.T1021C: p.W341R) in the MEN1 gene. The p.W341R mutation in the MEN1 gene showed complete co-segregation in the family. Whole exome capture and sequencing led to the discovery of a missense mutation in the MEN1 gene and ruling out of the additional candidates in a single experiment. The limited expressivity of this mutation may imply a specific genotype-phenotype correlation for this mutation.

MicroRNAs: fundamental regulators of gene expression in major affective disorders and suicidal behavior?

Abstract: Major affective disorders are one of the foremost causes of morbidity worldwide; such disabling conditions are also frequently associated with suicidal behavior (Innamorati et al., 2011; Gonda et al., 2012; Serafini et al., 2012). Although many psychopharmacological agents are currently available, in particular for the treatment of major depressive disorder (MDD) (Serafini et al., 2013), our knowledge concerning the molecular and cellular mechanisms underlying this complex condition is still limited. Indeed, even minor alterations in the expression of genes regulating neural and structural plasticity may be crucial to understanding the pathogenesis of major affective disorders (Dwivedi et al., 2009a,b; Serafini et al., 2011, 2012). MiRNAs are gene expression regulators critically affecting brain development that have been investigated as potential biomarkers for the diagnosis, management, treatment, and progression of neuropsychiatric disorders (Machado-Vieira et al., 2010; Saugstad, 2010; Dwivedi, 2011). Several facets of miRNA expression alterations are currently under investigation to gain insight into the pathology of neuronal disorders (Hansen et al., 2007; Lopez et al., 2013): miRNA expression alterations in pathophysiological models of disease (Ziu et al., 2011; Brandenburger et al., 2012); miRNA expression alterations in the blood of patients, most of which represent further downstream or compensatory effects (Schipper et al., 2007; Gallego et al., 2012); and miRNAs and their effectors acting as targets for the action of psychoactive drugs such as antidepressants and mood stabilizers (Zhou et al., 2009; Baudry et al., 2010; Oved et al., 2012). Several miRNAs have emerged as potential mediators of depressive pathophysiology. The existence of polymorphisms in pre-miR-30e (Xu et al., 2010) and pre-miR-182 (Saus et al., 2010) has been associated with an increased risk of major depression. Depressive behavioral responses have been induced by miR-16 up-regulation in the raphe nuclei and hippocampus, with the latter associated with subsequent down-regulation of BNDF (Bai et al., 2012). MiR-16 down-regulation within the locus coeruleus was also induced in depressive mouse models (Launay et al., 2011). Smalheiser et al. (2012) investigated the expression of miRNAs in the prefrontal cortex (specifically in Brodmann Area 9) of 18 antidepressant-free depressed suicide victims, and 17 well-matched non-psychiatric controls, whose information was collected using the psychological autopsy method. In this study, global miRNA expression was significantly down-regulated by 17%, and 24 miRNAs were down-regulated by at least 30%. The authors also found significant down-regulation in an extensive inter-connected network of 21 miRNAs involved in cellular growth and differentiation. Notably, both the global decrease of miRNA expression, as well as its decreased variability, are consistent with hypo-activation of the frontal cortex in depressed subjects. Interestingly, the noted miRNAs have been suggested to be down-regulated in the frontal cortex of rats treated with corticosterone, and therefore, might be crucial in regulating stress-mediated miRNA expression in depressed subjects (Dwivedi et al., unpublished data). Hence, alterations in miRNA expression may be a fundamental event underlying gene network reorganization associated with major depression. Nevertheless, a comprehensive understanding of miRNA networks dysregulated in major depression and induced by antidepressant medications as a function of brain region is currently unknown (Mouillet-Richard et al., 2012). It is also generally poorly understood how miRNA regulation affects cellular signaling networks in these biological processes. Here we provide an overview and critical review of the published work, particularly examining the role of miR-185 in major depression and suicidal behavior.

Meeting summary: Ethical aspects of whole exome and whole genome sequencing studies (WES/WGS) in rare diseases, Tel Aviv, Israel, January 2013.

Abstract: A recent E-Rare workshop reviewed the ethical aspects of whole exome and whole genome-sequencing studies (WES and WGS, respectively) in rare diseases. Leveraging new genomic technologies, which output vast amounts of known and novel genetic variants, researchers are learning more about the genetic basis and mechanisms involved in rare diseases. In some cases, these findings are translated into diagnostic tools for the benefit of rare disease patients. Among the disclosed data, which can assist in treatment management, incidental findings await, bringing with them ethical concerns for the clinicians, researchers and patients.

Assembly Algorithms for Deep Sequencing Data: Basics and Pitfalls

Abstract: Our ability to sequence the genomic data at our disposal is limited. At each experiment we can sequence reliably only a short fraction of even the smallest genome. We are then faced with the challenge of assembly-combining the short patches we have into a correct reconstruction of as large as possible a fragment of the original sample. The problem has been thoroughly researched and many commercial and academic tools exist to carry it out. However due to basic features of the problem the results of even our best efforts will be sometimes disappointing for the researcher. In this chapter we will try to explain why the assembly problem is so hard, what future directions may alleviate it in the near future, and what can be realistically expected from a current assembly experiment.

Genetics research: jumping into the deep end of the pool.

Abstract: The publication of the human genome, more than a decade ago, alongside the development of high-throughput technologies for DNA sequencing, marked the dawn of a new era in genetics. Large genomic projects have been initiated to decipher the mysteries hidden within the human genetic code. With the rapidly ever-growing amount of genetic information, and the importance of understanding what it all means, there is a need to generate an interdisciplinary hub that will connect researchers, both experimentalists and bioinformaticians, along with physicians and community representatives in order to develop a common genomic language. This should lead to an accessible, readable and interpretive human genome with a short list of personal actionable items. We will then be able to declare that we are moving ever closer to the point at which one's own genome will affect one's personal life at a scope beyond our current comprehension.