Dai dati grezzi all’interpretazione biologica: progettazione e analisi degli esperimenti di espressione genica realizzati mediante microarray

Negli ultimi venti anni la genetica e la biologia molecolare hanno contribuito significativamente al progresso scientifico-medico, fornendo strumenti per isolare, clonare e studiare molti dei geni che compongono il genoma umano. E’ ora possibile analizzare contemporaneamente l'espressione di migliaia di geni, ossia valutare quello che viene chiamato profilo genico, grazie all’uso di speciali supporti tecnologicamente avanzati denominati microarray. Un singolo esperimento di espressione genica realizzato con microarray produce migliaia di dati, per i quali è necessario un approccio rigoroso di tipo matematico e bioinformatico, sia nelle fasi di acquisizione e analisi che in quelle di interpretazione e archiviazione. A differenza delle fasi di preparazione dei campioni e ibridizzazione dei vetrini, che ormai sono regolate da protocolli sufficientemente standardizzati, i passaggi che portano dall’estrazione dei dati all’interpretazione biologica dei risultati non possono essere riassunti in un protocollo unico. Questo progetto di dottorato ha avuto lo scopo di studiare i metodi di progettazione di un esperimento di espressione genica mediante microarray e gli strumenti bioinformatici che servono a realizzare le fasi di estrazione e pre-trattamento dei dati, l’analisi statistica e l’interpretazione dei risultati. Tali metodi sono stati applicati a quattro esperimenti realizzati nel laboratorio presso il quale è stata svolta questa tesi. Sono stati individuati, fra quelli disponibili, i metodi bioinformatici per l’estrazione, il pre-trattamento e l’analisi statistica dei dati più affidabili e versatili per l’eliminazione degli errori legati alla metodica e per l’acquisizione di un dato statisticamente robusto. Il confronto critico dei metodi analizzati ha messo in luce la necessità di mettere a punto una soluzione ottimale di analisi per ciascun esperimento. La valutazione degli strumenti utili per l’interpretazione biologica dei risultati ha messo, invece, in evidenza profonde limitazioni legate essenzialmente all’assenza di informazioni ordinatamente catalogate e alla incompleta modellazione dei processi di co-regolazione genica nelle banche dati

Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

BACKGROUND: BRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer. METHODS: We compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain. RESULTS: 201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair. CONCLUSIONS: Our results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity

Genetically-Driven Enhancement of Dopaminergic Transmission Affects Moral Acceptability in Females but Not in Males: A Pilot Study

Moral behavior has been a key topic of debate for philosophy and psychology for a long time. In recent years, thanks to the development of novel methodologies in cognitive sciences, the question of how we make moral choices has expanded to the study of neurobiological correlates that subtend the mental processes involved in moral behavior. For instance, in vivo brain imaging studies have shown that distinct patterns of brain neural activity, associated with emotional response and cognitive processes, are involved in moral judgment. Moreover, while it is well-known that responses to the same moral dilemmas differ across individuals, to what extent this variability may be rooted in genetics still remains to be understood. As dopamine is a key modulator of neural processes underlying executive functions, we questioned whether genetic polymorphisms associated with decision-making and dopaminergic neurotransmission modulation would contribute to the observed variability in moral judgment. To this aim, we genotyped five genetic variants of the dopaminergic pathway [rs1800955 in the dopamine receptor D4 (DRD4) gene, DRD4 48 bp variable number of tandem repeat (VNTR), solute carrier family 6 member 3 (SLC6A3) 40 bp VNTR, rs4680 in the catechol-O-methyl transferase (COMT) gene, and rs1800497 in the ankyrin repeat and kinase domain containing 1 (ANKK1) gene] in 200 subjects, who were requested to answer 56 moral dilemmas. As these variants are all located in genes belonging to the dopaminergic pathway, they were combined in multilocus genetic profiles for the association analysis. While no individual variant showed any significant effects on moral dilemma responses, the multilocus genetic profile analysis revealed a significant gender-specific influence on human moral acceptability. Specifically, those genotype combinations that improve dopaminergic signaling selectively increased moral acceptability in females, by making their responses to moral dilemmas more similar to those provided by males. As females usually give more emotionally-based answers and engage the “emotional brain” more than males, our results, though preliminary and therefore in need of replication in independent samples, suggest that this increase in dopamine availability enhances the cognitive and reduces the emotional components of moral decision-making in females, thus favoring a more rationally-driven decision process

Analisi dei dati di espressione genica ottenuti mediante microarray: confronto critico tra più metodi statistici accettati.

Questa tesi ha avuto l’obiettivo di fare un’analisi critica di diversi approcci statistici utilizzati in bioinformatica per la selezione di geni differenzialmente espressi in esperimenti condotti mediante microarray a cDNA. Allo scopo di evidenziare le differenze metodologiche fra i criteri messi a confronto, è stata realizzata una validazione informatica incrociata degli approcci statistici, avvalendosi dei dati pubblicati in uno studio di identificazione di geni coinvolti nei processi di crescita e invecchiamento dei nematodi

Gene by gender interplay in moral choices

While philosophers, psychologists and cognitive scientists have proposed distinct definitions of moral judgment, recent studies suggest that moral choices are modulated by neurobiological mechanisms. The pioneering works by Greene showed that certain brain areas may be considered “specific” for moral decision and provided support for a dual-process theory, according to which two different patterns of neural activity are involved in moral choices: a fast, unconscious "emotional" system, and a slow, conscious "cognitive" system1, 2, 3. Furthermore, genetic associations between two allelic variants in serotonin transporter and oxytocin receptor genes, and moral judgment have been reported4, 5. Because of their described association with impulsive behavior6, 7, 8, 9, we questioned whether four polymorphisms in genes involved in serotonergic and dopaminergic neurotransmission (SLC6A3–VNTR, DRD4-VNTR, DRD4 rs1800955, COMT rs4680) would modulate the cognitive and emotional processes at the basis of controversial moral choices. After signing an informed consent, 200 (102F) University students were recruited in a moral dilemma paradigm (N=56) designed to assess three variables: moral action type (Means vs Side Effect), life expectancy (Normal vs Reduced), self-involvement (Involvement vs Non-Involvement). They also provided saliva samples for DNA collection and completed the Impulsivity-Venturesomeness-Empathy Questionnaire (I7). Significant differences between males and females were observed in the I7 scale scores. Moreover, only in males Venturesomeness scores correlated with the number of utilitarian responses. Males, compared to females, gave a higher number of utilitarian responses, showed longer response times for non-utilitarian answers and judged as more acceptable the endorsed moral actions. Interestingly, only females showed a significant association between allelic variants involved in dopamine level regulation in striatum and prefrontal cortex, and moral choices. Our results are the first ones showing that impulsivity and genetic profile influence moral judgment in a gender-related manner, thus shedding new light on the neurobiological mechanisms underlying moral choices. BIBLIOGRAPHY 1. Greene JD, Sommerville RB, Nystrom LE, Darley JM, Cohen JD (2001). An fMRI investigation of emotional engagement in moral judgment. Science 293(5537): 2105–2108. 2. Greene JD, Nystrom LE, Engell AD, Darley JM, Cohen JD (2004). The neural bases of cognitive conflict and control in moral judgment. Neuron 44(2): 389–400. 3. Greene JD (2009). Dual-process morality and the personal/impersonal distinction: a reply to McGuire, Langdon, Coltheart, and Mackenzie. J Exp Soc Psychol 45(3): 581-584. 4. Marsh AA, Crowe SL, Yu HH, Gorodetsky EK, Goldman D, Blair RJR (2011). Serotonin transporter genotype (5-HTTLPR) predicts utilitarian moral judgments. Plos One 6(10): e25148. 5. Walter NT, Montag C, Markett S, Felten A, Voigt G, Reuter M (2012). Ignorance is no excuse: moral judgments are influenced by a genetic variation on the oxytocin receptor gene. Brain and Cognition 78(3): 268-273. 6. Munafò MR, Yalcin B, Willis-Owen SA, Flint J (2008). Association of the dopamine D4 receptor (DRD4) gene and approach-related personality traits: meta-analysis and new data. Biol Psychiatry 63(2): 197-206. 7. Joyce PR, McHugh PC, Light KJ, Rowe S, Miller AL, Kennedy MA (2009). Relationships between angry-impulsive personality traits and genetic polymorphisms of the dopamine transporter. Biol Psychiatry 66(8): 717-721. 8. Reiner I, Spangler G (2011). Dopamine D4 receptor exon III polymorphism, adverse life events and personality traits in a nonclinical German adult sample. Neuropsychobiology 63(1): 52-58. 9. Soeiro-De-Souza MG, Stanford MS, Bio DS, Machado-Vieira R, Moreno RA (2013). Association of the COMT Met158 allele with trait impulsivity in healthy young adult. Mol Med Rep 7(4): 1067-1072

Interpreting the gene expression microarray results: a user-based experience.

In recent years many tools have been developed to cope with the interpretation of gene expression results from microarray experiments. The effectiveness of these tools largely depends on their ease of use by biomedical researchers. Tools based on effective computational methods, indeed, cannot be fully exploited by users if they are not supported by an intuitive interface, a large set of utilities and effective outputs. In this paper, ten tools for the interpretation of gene expression microarray results have been tested on eleven microarray datasets and evaluated according to eight assessment criteria: 1. interface design and usability, 2. easiness of input submission, 3. effectiveness of output representation, 4. efficacy of the downloaded outputs, 5. possibility to submit multiple gene IDs, 6. sources of information, 7. provision of different statistical tests and 8. supply of multiple test correction methods. Strengths and weaknesses of each tool are highlighted: a. to provide useful tips to users dealing with the biological interpretation of microarray results; b. to draw the attention of software developers on the usability of their tools

Comparison of the transcriptional response to T₁AM with the known genomic effects of thyroid hormone, Insulin and Cortisol (up-regulated = ↑, down-regulated = ↓, not regulated or no data available = -).

<p>Comparison of the transcriptional response to T₁AM with the known genomic effects of thyroid hormone, Insulin and Cortisol (up-regulated  = ↑, down-regulated  = ↓, not regulated or no data available  =  -).</p

Differentially expressed genes in the subcutaneous adipose tissue annotated by <i>Onto-Express</i>, <i>GeneCards</i> and <i>COREMINE</i>.

<p>Differentially expressed genes in the subcutaneous adipose tissue annotated by <i>Onto-Express</i>, <i>GeneCards</i> and <i>COREMINE</i>.</p

Changes in gene expression evidenced by microarrays were confirmed by RT-qPCR for four of the five genes tested in the subcutaneus adipose tissue (the differential expression of Cebpb was not statistically significant) and for all the seven genes tested in the liver.

<p>Data reported as: log2 fold-change ± SE; *: p≤0.05.</p