Gene Expression Analysis Methods on Microarray Data a A Review

In recent years a new type of experiments are changing the way that biologists and other specialists analyze many problems. These are called high throughput experiments and the main difference with those that were performed some years ago is mainly in the quantity of the data obtained from them. Thanks to the technology known generically as microarrays, it is possible to study nowadays in a single experiment the behavior of all the genes of an organism under different conditions. The data generated by these experiments may consist from thousands to millions of variables and they pose many challenges to the scientists who have to analyze them. Many of these are of statistical nature and will be the center of this review. There are many types of microarrays which have been developed to answer different biological questions and some of them will be explained later. For the sake of simplicity we start with the most well known ones: expression microarrays

Rhythmic dynamics and synchronization via dimensionality reduction : application to human gait

Reliable characterization of locomotor dynamics of human walking is vital to understanding the neuromuscular control of human locomotion and disease diagnosis. However, the inherent oscillation and ubiquity of noise in such non-strictly periodic signals pose great challenges to current methodologies. To this end, we exploit the state-of-the-art technology in pattern recognition and, specifically, dimensionality reduction techniques, and propose to reconstruct and characterize the dynamics accurately on the cycle scale of the signal. This is achieved by deriving a low-dimensional representation of the cycles through global optimization, which effectively preserves the topology of the cycles that are embedded in a high-dimensional Euclidian space. Our approach demonstrates a clear advantage in capturing the intrinsic dynamics and probing the subtle synchronization patterns from uni/bivariate oscillatory signals over traditional methods. Application to human gait data for healthy subjects and diabetics reveals a significant difference in the dynamics of ankle movements and ankle-knee coordination, but not in knee movements. These results indicate that the impaired sensory feedback from the feet due to diabetes does not influence the knee movement in general, and that normal human walking is not critically dependent on the feedback from the peripheral nervous system

Gene Expression Response in Early Developmental Stages of Rainbow Trout Exposed to Ecologically Relevant Concentrations of Malathion

Understanding the early life stage toxic effects of environmental organophosphate exposure on organism health is crucial to identifying biomarkers that can be used for preventative care. Malathion, a potent organophosphate, is one of the most widely used organophosphates in agriculture and pest eradication. Due to its widespread use, pesticide runoff into area bodies of water poses a great threat to aquatic life and human inhabitants. Acute exposure to high concentrations of malathion causes neurological abnormalities and can result in respiratory failure, muscle spasms, and mental confusion in humans. In the present study, the effects of malathion are observed following acute, low-level exposure; however, most diagnostic tests require sustained exposure to concentrations high enough to induce acetylcholinesterase inhibition. There is a direct relationship between AChE inhibition and acute exposure to malathion, that results in systematic disturbances in neural function and elicits overt toxicity. While disrupted AChE activity serves as a biomarker, it is not sensitive to low levels of malathion exposure. Therefore, it is necessary to identify novel biomarkers that are more sensitive to malathion exposure so that steps can be taken to ensure the safety of humans and aquatic life before the neurological complications can develop. Here were we report the discovery of two new potential biomarkers that are expressed following acute, low-concentration malathion exposure. We used two early life stages of rainbow trout (sac fry and swim-up fry) exposed to varying malathion levels (3-100 parts per billion, ppb) over 48 hours. At these levels, swim-up fry had heightened mortality rates compared to sac fry, indicating that they may exhibit change in gene expression. To identify transcriptional biomarkers, a 16K salmonid cDNA microarray was used; 349 genes were found to be differentially expressed at concentrations as low as 10 ppb. Additionally, we further analyzed malathion responsive genes using qPCR, network and ontologic analysis. The results from qPCR revealed that the gene encoding for a small heat shock protein, HSP30, was robustly upregulated. A second gene put forth as a candidate biomarker in this study is cytochrome P4501A3 (CYP1A3); this study also shows paralog CYP1A1 was not found to be malathion inducible in either life stage. Network and ontologic analysis suggested changes in expression of genes involved in metal ion binding, catalytic activity, transport, oxidation-reduction, metabolism and stress response. The novel findings of this study contribute to the construction of a repertoire of predictive biomarkers, induced by malathion exposure, that may also be used as tools to survey fish population health

Identification and Functional Assessment of Novel Neuromuscular Disease-Causing Genes

Inherited neuromuscular diseases comprise a highly heterogeneous group of disorders characterized by the impairment of the neural structures or motor unit components responsible for the generation of movement. While as single gene-associated disorder the majority of them are rare, taken together their estimated prevalence reaches 1 – 3 cases / 1000 individuals. Due to their elevated morbidity and mortality, they represent a significant health burden for the affected individuals, their families, and the healthcare systems. Moreover, their clinical and genetic heterogeneity makes their diagnosis a long and complex process, which often requires specialized diagnostic procedures and poses a challenge in about half of the cases. However, thanks to decreasing costs and increased availability of next-generation sequencing technologies, the last years had witnessed a rise in the number of novel genes associated to neuromuscular disorders. In this study, we identified three novel neuromuscular disease-causing genes: PIEZO2, whose biallelic loss-of-function mutations cause distal arthrogryposis with impaired proprioception and touch; VAMP1, whose biallelic loss-of-function mutations cause a novel presynaptic congenital myasthenic syndrome; CAPRIN1, whose specific p.Pro512Leu mutation causes a neurodegenerative disorder characterized by ataxia and muscle weakness. For PIEZO2, we identified biallelic loss-of-function mutations using exome sequencing, SNPchip-based linkage analysis, DNA microarray, and Sanger sequencing in ten affected individuals of four independent families showing arthrogryposis, hypotonia, respiratory insufficiency at birth, scoliosis, and delayed motor development. This phenotype is clearly distinct from distal arthrogryposis with ocular anomalies which characterize the autosomal dominant distal arthrogryposis 3 (DA3), distal arthrogryposis 5 (DA5), and Marden-Walker syndrome (MWKS). While these disorders are caused by heterozygous gain-of-function mutations in PIEZO2, the novel reported mutations result in the loss of PIEZO2, since they lead to nonsense-mediated mRNA decay in patient-derived fibroblast cell lines. PIEZO2 is a mechanosensitive ion channel playing a major role in light-touch sensation and proprioception. Mice ubiquitously depleted of PIEZO2 die postnatally because of respiratory distress, while individuals lacking PIEZO2 develop a neuromuscular disorder, likely due to the loss of proprioception inputs in muscles. For VAMP1, we identified biallelic loss-of-function mutations using exome or genome sequencing in two pairs of siblings from two independent families affected by a novel congenital myasthenic syndrome. Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. The two described homozygous mutations are a frameshift and a missense mutation of a highly conserved residue, therefore are likely to result in the loss of VAMP1 function. Indeed, the phenotype is resembled by VAMP1lew/lew mice, which carry a homozygous VAMP1 truncating mutation and show neurophysiological features of presynaptic impairment. For CAPRIN1, we identified the identical de novo c.1535C>T (p.Pro512Leu) missense variant using trio exome sequencing in two unrelated individuals displaying early-onset ataxia, dysarthria, cognitive decline and muscle weakness. This mutation causes the substitution of a highly conserved residue and in silico tools predict an increase in the protein aggregation propensity. Overexpression of CAPRIN1-P512L caused the formation of insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders, such as ATXN2, GEMIN5, SNRNP200, and SNCA. Upon differentiation in cortical neurons of induced pluripotent stem cell (iPSC) lines where the CAPRIN1-P512L was introduced via CRISPR/Cas9, reduced neuronal activity and altered stress granules dynamics were observed in the lines harboring the mutation. Moreover, nano-differential scanning fluorimetry revealed that CAPRIN1-P512L adopts an extended conformation, and fluorescence microscopy demonstrated that RNA greatly enhances its aggregation in vitro. Taken together, this study associates: (1) biallelic loss-of-function mutations in PIEZO2 with the autosomal recessive distal arthrogryposis with impaired proprioception and touch; (2) biallelic loss-of-function mutations in VAMP1 with an autosomal recessive presynaptic congenital myasthenic syndrome; (3) a recurrent de novo p.Pro512Leu mutation of CAPRIN1 with a neurodegenerative disorder characterized by ataxia and muscle weakness

Treatment-resistant ophthalmoplegia in myasthenia gravis: Clinical, molecular and functional studies of patient-derived orbital tissues

Introduction: Myasthenia gravis (MG) is an immune-mediated disorder affecting the neuromuscular junction. Weakness of the extraocular muscles (EOMs) occurs frequently in MG and typically responds to immune therapies similarly to the non-ocular muscles. Susceptible individuals with the ophthalmoplegic subphenotype of MG (OP-MG), which occurs almost exclusively in acetylcholine receptor positive MG (AChR-MG), may manifest treatmentresistant extraocular muscle weakness despite the use of standard immune therapies. The pathogenetic mechanisms involved in the development of treatment-resistant ophthalmoplegia in MG are still unknown and no effective treatment currently exists. Aim: To investigate the molecular-genetic pathogenesis of the OP-MG subphenotype. Methods: Triangulation of data from clinical observations, review of MG muscle biopsy histopathology, gene expression studies in OP-MG patient-derived orbital muscles (AChR-MG) and bioenergetic studies in highly specialised perimysial ocular fibroblasts of these OP-MG cases was used to identify the underlying pathogenetic mechanisms of OP-MG and to verify previous hypotheses generated by next generation sequencing studies. Results: Myasthenic ophthalmoparesis may persist despite immune therapies in 40% of cases in the first year of immune treatment. Delay to diagnosis of MG and therefore initiation of treatment (>1 year) was an unfavourable prognostic factor for resolution of ophthalmoparesis and suggested that with prolonged weakness, pathological changes may occur at the level of the muscle. Review of the literature documenting histopathology in MG muscle biopsies showed that neurogenic atrophy and features of mitochondrial stress, which may be secondary consequences of functional denervation and reduced contractility, are frequently observed in MG muscle biopsies and the EOMs may be particularly susceptible, demonstrating features of fatty and fibrocellular replacement of myofibres. Gene expression studies performed in the orbital muscles of OP-MG and non-MG control cases supported the hypotheses of previous unbiased genomic studies showing that genes harbouring OP-MG associated gene variants may be involved in a dysregulated network of genes including genes in pathways involved in atrophy signalling, muscle contractility and mitochondrial homeostasis. Several genes were significantly downregulated in the OP-MG orbital muscles compared with controls. MicroRNAs which are biological regulators of gene expression, were hypothesized to be a potential pathogenetic mechanism causing downregulation of these genes in OP-MG orbital muscles and several microRNAs highly expressed in EOMs were associated with the significantly repressed genes in OP-MG orbital muscle using available data in public microRNA databases. Preliminary dynamic bioenergetic assays in perimysial ocular fibroblasts derived from the EOM myotendons of OP-MG and non-MG control cases suggested that regulation of mitochondrial homeostasis may be altered in the context of MG. Conclusion: Gene expression analyses in patient-derived orbital muscles support the hypotheses of previous genomic studies suggesting that pathogenetic mechanisms involving pathways relating to muscle atrophy, contractility and mitochondrial homeostasis may by triggered in the EOMs in the context of MG. Dysregulation of these pathways is likely to impact EOM regeneration in the context of MG-induced complement-mediated attack as well as contractility in this specialized muscle allotype with a high firing rate. These complex aberrant molecular-genetic interactions may contribute to persistent ophthalmoplegia despite adequate immune therapies in OP-MG cases

Gene expression studies of pregastrulation development: the basement membrane is essential for cell differentiation

Basement membranes (BMs) are sheet-like structures of extracellular matrix. They act as a supporting structure but can also significantly influence cellular behavior in development, tissue homeostasis and disease. Laminins, a major BM component, are multidomain proteins, consisting of three polypeptide chains (α, β and γ). During pregastrulation development, stem cells convert and epithelial tissues are formed. This process is faithfully mimicked in vitro by embryoid body (EB) cultures. Fibroblast growth factor (FGF) signaling is crucial when the step-like process of EB development is initiated with the formation of an endoderm. A subendodermal BM is formed, in which the globular domains LG4-5 of the laminin α1 chain (α1LG4-5) are responsible for the induction of the epiblast EBs derived form embryonic stem (ES) cells, modified to repress FGF receptor signaling, have been described before. However, a full-scale analysis of the transcriptome was missing. We therefore analysed these EBs at four time points during differentiation by the use of microarray technique. An extensive catalogue of affected genes was reported. A majority of the genes directed by FGF signalling were encoding BM and endodermal proteins. In addition, we also analysed the expression profile of wild type EBs. In both these studies, we found interesting genes not previously described in early development or identified as FGF targets. Hopefully, our gene catalogue will be a valuable source for the scientific community interested in FGF signaling, developmental biology and stem cell research. Furthermore, a gene expression study was set up to get a better insight of epiblast inducement by α1LG4-5. EBs derived form ES cells with a targeted deletion of the α1LG4-5 domains were analysed. To our surprise, we found several indications of an incomplete differentiation of the visceral endoderm. We therefore hypothesize a novel autocrine mechanism for α1LG4-5 in regulating the developing endoderm. We also suggest novel roles for laminin LG4-5 in the neuromuscular system. Using laminin α2 chain deficient mice overexpressing laminin α1 chain lacking the LG4-5 domains, we show that these domains, and consequently binding to the receptor dystroglycan are not crucial in diaphragm and heart, but essential in the peripheral nervous system

Developing RNA diagnostics for studying healthy human ageing

Developing strategies to cope with increase in the ageing population and age-related chronic diseases is one of the societies biggest challenges. The characteristics of the ageing process shows significant inter-individual variation. Building genomic signatures that could account for variation in health outcomes with age may facilitate early prognosis of individual age-correlated diseases (e.g. cancer, coronary artery diseases and dementia) and help in developing better targeted treatments provided years in advance of acquiring disabling symptoms for these diseases. The aim of this thesis was to explore methods for diagnosing molecular features of human ageing. In particular, we utilise multi-platform transcriptomics, independent clinical data and classification methods to evaluate which human tissues demonstrate a reproducible molecular signature for age and which clinical phenotypes correlated with these new RNA biomarkers. [Continues.

The role of genetic variation in selected human musculoskeletal ageing traits.

Loss of muscle mass and function, termed sarcopenia, occurs commonly with advancing age. This loss of strength can have a profound impacts on an individual’s life expectancy and quality of life. Population genetic studies can provide information on underlying biological mechanisms, but little was known about the genetic contributions to sarcopenia. By using data from multi-national community based studies of 256,523 individuals of European ancestry aged 60 years or older I have identified 15 genomic risk loci for muscle weakness with age. I have shown that the genetic contributions to muscle weakness in later life have novel characteristics not seen in studies of muscle strength at younger ages. I have also shown that for a section of the older population meeting the criteria for sarcopenia, there is a substantial auto-immune component separate from diagnosed autoimmune conditions, such as Rheumatoid arthritis. Analysis of sex-specific cohorts has highlighted that the underlying genetics contributing to muscle weakness with age differ between the sexes. Additional research on the shared pathways between age-related traits and muscle weakness with age has shown that diabetes, rheumatoid arthritis and life courses traits, for example birth weight, share at least some of the same biological pathways. Biological pathways implicated included transcription regulation, processing of misfolded proteins, cell growth and development. In conclusion I have identified several common genetic variants associated with sarcopenia in humans, which has highlighted an autoimmune component and several shared casual pathways with traits ranging from life-course and growth traits through to later life conditions such as Rheumatoid arthritis and diabetes. These findings should inform efforts to prevent and treat muscle loss with advancing age, and may more personalised approaches to intervention

Computational Methods for the Identification of Statistically Significant Genes: Applications to Gene Expression Data of Various Human Diseases

Σε αυτή την διατριβή αντιμετωπίσαμε το πρόβλημα της επιλογής γονιδίων από ταξινομημένες λίστες. Προτείναμε μια νέα υβριδική μέθοδο επιλογής χαρακτηριστικών (mAP-KL) που συνδυάζει με επιτυχία μια μέθοδο πολλαπλού ελέγχου υποθέσεων και μια μέθοδο συσταδοποίησης (Affinity Propagation) μαζί με έναν δείκτη ποιότητας συστάδων των Krzanowski & Lai, για την επιλογή ενός μικρού αλλά αντιπροσωπευτικού υποσυνόλου γονιδίων. Υποβάλαμε τη μέθοδό μας σε διάφορες αξιολογήσεις με δεδομένα προσομοίωσης μικροσυστοιχιών καθώς και με πραγματικά δεδομένα μικροσυστοιχιών. Τα συνολικά αποτελέσματα της αξιολόγησης δείχνουν ότι η mAP-KL παράγει συνοπτικά υποσύνολά από -υπογραφές γονιδιακής έκφρασης οι οποίες σχετίζονται βιολογικά και μπορούν να χρησιμεύσουν ως ένα πολύτιμο διακριτικό εργαλείο για διαγνωστικούς και προγνωστικούς σκοπούς, με τον εντοπισμό πιθανών βιοδεικτών της νόσου σε ένα ευρύ φάσμα ασθενειών. Τέλος, προκειμένου να δώσουμε στην ερευνητική κοινότητα με τη δυνατότητα να εφαρμόσει την mAP-KL σε οποιοδήποτε σύνολο δεδομένων γονιδιακής έκφρασης, αναπτύξαμε τη μεθοδολογία μας σε ένα Bioconductor/R- πακέτο το οποίο συνοδεύεται και από άλλες επιπλέον λειτουργίες.In this dissertation, we address the problem of gene selection from ranked gene lists. We propose a new hybrid feature selection method (mAP-KL) that combines successfully multiple hypothesis testing and affinity propagation clustering algorithm along with the Krzanowski & Lai cluster quality index, to select a small yet informative subset of genes. We subject our method across a variety of validation tests on simulated microarray data as well as on real microarray data. The overall evaluation results suggest that mAP-KL generates concise yet biologically relevant and informative n-gene expression signatures, which can serve as a valuable discrimination tool for diagnostic and prognostic purposes, by identifying potential disease biomarkers in a broad range of diseases. Finally, to provide the research community with the capability to apply mAP-KL in any given gene expression dataset, we have implemented this methodology to a Bioconductor/R-package accompanied with extra functionalities