Analisi di “Copy Number Variants” ed identificazione di nuovi geni candidati per l’Autismo e Ritardo Mentale

I disturbi dello spettro autistico (DSA) ed il ritardo mentale (RM) sono caratterizzati da un’eziologia genetica complessa ed eterogenea. Grazie ai recenti sviluppi nella ricerca genomica, è stato possibile dimostrare il ruolo di numerose copy number variants (CNVs) nella patogenesi di questi disturbi, anche se nella maggior parte dei casi l’eziologia rimane ancora sconosciuta. Questo lavoro riguarda l’identificazione e la caratterizzazione dei CNVs in famiglie con DSA e RM. E’ stata studiata una microdelezione in 7q31 che coinvolge i geni IMMP2L e DOCK4, trasmessa dalla madre con dislessia a due figli con autismo ed una figlia con dislessia. Nella stessa famiglia segrega una seconda microdelezione in 2q14 che inattiva il gene CNTNAP5 ed è trasmessa dal padre (con tratti autistici) ai due figli con autismo. Abbiamo quindi ipotizzato che i geni DOCK4 e CNTNAP5 potessero essere implicati, rispettivamente, nella suscettibilità a dislessia e DSA. Lo screening di numerosi individui affetti ha supportato la nostra ipotesi, con l’identificazione di una nuova microdelezione di DOCK4 che segrega con la dislessia, e 3 nuove varianti missenso in CNTNAP5 in individui con autismo. Dall’analisi genomica comparativa su array (aCGH) di individui con RM, è stata identificata una delezione nella regione 7q31.32, che coinvolge il gene CADPS2, in due fratelli con RM e tratti autistici, probabilmente ereditata dalla madre. Lo screening di mutazione di questo gene in individui con autismo o RM, ha portato all’identificazione di 3 varianti non sinonime, assenti nei controlli, ed ereditate per via materna. Poiché CADPS2 risiede in una regione genomica che contiene loci soggetti ad imprinting, abbiamo ipotizzato che il gene CADPS2 possa essere anch’esso caratterizzato da imprinting, con espressione monoallelica materna. Lo studio di espressione di CADPS2 in cellule del sangue ha avvalorato questa ipotesi, implicando perciò CADPS2 come un nuovo gene di suscettibilità per il RM e DSA.

New techniques for the remote sensing of foliar nitrogen concentration in forest ecosystems.

During my Doctoral study I researched about the remote detection of canopy N concentration in forest stands, its potentials and problems, under many overlapping perspectives. The study consisted of three parts. In S. Rossore 2000 dataset analysis, I tested regressions between N concentration and NIR reflectances derived from different sources (field samples, airborne and satellite sensors). The analysis was further expanded using a larger dataset acquired in year 2009 as part of a new campaign funded by the ESA. In both cases, a good correlation was observed between Landsat NIR, using both TM (2009) and ETM+ (2000) imagery, and N concentration measured by a CHN elemental analyzer. Concerning airborne sensors I did not obtain the same good results, mainly because of the large FOV of the two instruments, and to the anisotropy of vegetation reflectance. We also tested the relation between ground based ASD measures and nitrogen concentration, obtaining really good results. Thus, I decided to expand my study to the regional level, focusing only on field and satellite measures. I analyzed a large dataset for the whole of Catalonia, Spain; MODIS imagery was used, in consideration of its spectral characteristics and despite its rather poor spatial resolution. Also in this case a regression between nitrogen concentration and reflectances was found, but not so good as in previous experiences. Moreover, vegetation type was found to play an important role in the observed relationship. We concluded that MODIS is not the most suitable satellite sensor in realities like Italy and Catalonia, which present a patchy and inhomogeneous vegetation cover; so it could be utilized for the parameterization of eco-physiological and biogeochemical models, but not for really local nitrogen estimate. Thus multispectral sensors similar to Landsat Thematic Mapper, with better spatial resolution, could be the most appropriate sensors to estimate N concentration.

New computational biology tools for the systematic analysis of the structure and expression of human genes

From the late 1980s, the automation of sequencing techniques and the computer spread gave rise to a flourishing number of new molecular structures and sequences and to proliferation of new databases in which to store them. Here are presented three computational approaches able to analyse the massive amount of publicly avalilable data in order to answer to important biological questions. The first strategy studies the incorrect assignment of the first AUG codon in a messenger RNA (mRNA), due to the incomplete determination of its 5' end sequence. An extension of the mRNA 5' coding region was identified in 477 in human loci, out of all human known mRNAs analysed, using an automated expressed sequence tag (EST)-based approach. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 and the consequences for the functional studies are discussed. The second approach analyses the codon bias, the phenomenon in which distinct synonymous codons are used with different frequencies, and, following integration with a gene expression profile, estimates the total number of codons present across all the expressed mRNAs (named here "codonome value") in a given biological condition. Systematic analyses across different pathological and normal human tissues and multiple species shows a surprisingly tight correlation between the codon bias and the codonome bias. The third approach is useful to studies the expression of human autism spectrum disorder (ASD) implicated genes. ASD implicated genes sharing microRNA response elements (MREs) for the same microRNA are co-expressed in brain samples from healthy and ASD affected individuals. The different expression of a recently identified long non coding RNA which have four MREs for the same microRNA could disrupt the equilibrium in this network, but further analyses and experiments are needed.

Development, degeneration and neural network of the bodily self

The question addressed by this dissertation is how the human brain builds a coherent representation of the body, and how this representation is used to recognize its own body. Recent approaches by neuroimaging and TMS revealed hints for a distinct brain representation of human body, as compared with other stimulus categories. Neuropsychological studies demonstrated that body-parts and self body-parts recognition are separate processes sub-served by two different, even if possibly overlapping, networks within the brain. Bodily self-recognition is one aspect of our ability to distinguish between self and others and the self/other distinction is a crucial aspect of social behaviour. This is the reason why I have conducted a series of experiment on subjects with everyday difficulties in social and emotional behaviour, such as patients with autism spectrum disorders (ASD) and patients with Parkinson’s disease (PD). More specifically, I studied the implicit self body/face recognition (Chapter 6) and the influence of emotional body postures on bodily self-processing in TD children as well as in ASD children (Chapter 7). I found that the bodily self-recognition is present in TD and in ASD children and that emotional body postures modulate self and others’ body processing. Subsequently, I compared implicit and explicit bodily self-recognition in a neuro-degenerative pathology, such as in PD patients, and I found a selective deficit in implicit but not in explicit self-recognition (Chapter 8). This finding suggests that implicit and explicit bodily self-recognition are separate processes subtended by different mechanisms that can be selectively impaired. If the bodily self is crucial for self/other distinction, the space around the body (personal space) represents the space of interaction and communication with others. When, I studied this space in autism, I found that personal space regulation is impaired in ASD children (Chapter 9).

Investigating the role of Copy Number Variants in Specific Language Impairment and identification of new candidate genes

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

Characterization of the Caspr2 and NLGN2 ligands: a proteomic and biochemical approach

Autism Spectrum Disorder (ASD) is a range of early-onset conditions classified as neurodevelopmental disorders, characterized by deficits in social interactions and communication, as well as by restricted interest and repetitive behaviors. Among the proteins associated with this spectrum of disease there are Caspr2, α-NRXN1, NLGN1-4. Caspr2 is involved in the clustering of K+ channels at the juxtaparanodes, where it is proposed to bind TAG-1. Recent works reported a synaptic localization of Caspr2, but little is know on its role in this compartment. NRXNs and their ligand NLGNs, instead, have a well-defined role in the formation and maintenance of synapses. Among the neuroligins, NLGN2 binds NRXNs with the lowest affinity, suggesting that it could have other not yet characterized ligands. The aim of this work was to better characterize the binding of Caspr2 to TAG-1 and to identify new potential binding partner for Caspr2 and NLGN2. Unexpectedly, using Isothermal Titration Calorimetry and co-immunoprecipitation experiments the direct association of the first two proteins could not be verified and the results indicate that the first evidences reporting it were biased by false-positive artifacts. These findings, together with the uncharacterized synaptic localization of Caspr2, made the identification of new potential binding partners for this protein necessary. To find new proteins that associate with Caspr2 and NLGN2, affinity chromatography in tandem with mass spectrometry experiments were performed. Interestingly, about 25 new potential partners were found for these two proteins and NLGN1, that was originally included as a control: 5 of those, namely SFRP1, CLU, APOE, CNTN1 and TNR, were selected for further investigations. Only the association of CLU to NLGN2 was confirmed. In the future, screenings of the remaining candidates have to be carried out and the functional role for the proposed NLGN2-CLU complex has to be studied.

Spagnolo Tecnico Semplificato

L’obiettivo della presente dissertazione è quello di creare un nuovo linguaggio controllato, denominato Español Técnico Simplificado (ETS). Basato sulla specifica tecnica del Simplified Technical English (STE), ufficialmente conosciuta come ASD-STE100, lo spagnolo controllato ETS si presenta come un documento metalinguistico in grado di fornire ad un redattore o traduttore tecnico alcune regole specifiche per produrre un documento tecnico. La strategia di implementazione conduce allo studio preliminare di alcuni linguaggi controllati simili all’inglese STE, quali il Français Rationalisé e il Simplified Technical Spanish. Attraverso un approccio caratteristico della linguistica dei corpora, la soluzione proposta fornisce il nuovo linguaggio controllato mediante l’estrazione di informazioni specifiche da un corpus ad-hoc di lingua spagnola appositamente creato ed interrogato. I risultati evidenziano un metodo linguistico (controllato) utile a produrre documentazione tecnica priva di ogni eventuale ambiguità. Il sistema ETS, infatti, si fonda sul concetto della intelligibilità in quanto condizione necessaria da soddisfare nell’ambito della produzione di un testo controllato. E, attraverso la sua macrostruttura, il documento ETS fornisce gli strumenti necessari per rendere il testo controllato univoco. Infatti, tale struttura bipartita suddivide in maniera logica i dettami: una prima parte riguarda e contiene regole sintattiche e stilistiche; una seconda parte riguarda e contiene un dizionario di un numero limitato di lemmi opportunamente selezionati. Il tutto a favore del principio della biunivocità dei segni, in questo caso, della lingua spagnola. Il progetto, nel suo insieme, apre le porte ad un linguaggio nuovo in alternativa a quelli presenti, totalmente creato in accademia, che vale come prototipo a cui far seguire altri progetti di ricerca.

Analysis of Copy Number Variants identifies new candidate genes for Autism Spectrum Disorder and Intellectual Disability

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

Non Invasive Tools for Early Detection of Autism Spectrum Disorders

Autism Spectrum Disorders (ASDs) describe a set of neurodevelopmental disorders. ASD represents a significant public health problem. Currently, ASDs are not diagnosed before the 2nd year of life but an early identification of ASDs would be crucial as interventions are much more effective than specific therapies starting in later childhood. To this aim, cheap an contact-less automatic approaches recently aroused great clinical interest. Among them, the cry and the movements of the newborn, both involving the central nervous system, are proposed as possible indicators of neurological disorders. This PhD work is a first step towards solving this challenging problem. An integrated system is presented enabling the recording of audio (crying) and video (movements) data of the newborn, their automatic analysis with innovative techniques for the extraction of clinically relevant parameters and their classification with data mining techniques. New robust algorithms were developed for the selection of the voiced parts of the cry signal, the estimation of acoustic parameters based on the wavelet transform and the analysis of the infant’s general movements (GMs) through a new body model for segmentation and 2D reconstruction. In addition to a thorough literature review this thesis presents the state of the art on these topics that shows that no studies exist concerning normative ranges for newborn infant cry in the first 6 months of life nor the correlation between cry and movements. Through the new automatic methods a population of control infants (“low-risk”, LR) was compared to a group of “high-risk” (HR) infants, i.e. siblings of children already diagnosed with ASD. A subset of LR infants clinically diagnosed as newborns with Typical Development (TD) and one affected by ASD were compared. The results show that the selected acoustic parameters allow good differentiation between the two groups. This result provides new perspectives both diagnostic and therapeutic.

Sviluppo e caratterizzazione microstrutturale e tribologica di rivestimenti su lega di titanio per applicazioni biomedicali

Oggetto dello studio è stato lo sviluppo di rivestimenti con tecnica PEO (Plasma Electrolityc Ossidation) sulla lega di titanio Ti-6Al-4V, al fine di utilizzare questo materiale in sostituzione della lega CrCoMo nelle protesi d'anca e di ginocchio. Queste, ad oggi, sono le protesi articolari più diffuse e devono garantire contemporaneamente elevate prestazioni meccaniche (in particolare resistenza ad usura), affidabilità e biocompatibilità. La lega CrCoMo negli anni si è affermata nel campo protesico poiché è un materiale metallico avente elevata rigidezza abbinata a una buona resistenza a corrosione ed all'usura durante il movimento articolare. Un problema rilevante e frequente di questa lega è l'allergia di alcuni pazienti agli elementi di lega che la costituiscono o il rischio per i pazienti non allergici di subire un'ipersensibilizzazione, con conseguente sviluppo dell'allergia e necessità di sostituire la protesi. La lega Ti-6Al-4V potrebbe essere una valida alternativa data la sua elevata biocompatibilità e le sue proprietà meccaniche, tanto che è già ampiamente utilizzata nella costruzione di protesi statiche come chiodi o viti. Purtroppo ad oggi non è stato possibile l'utilizzo di questa negli accoppiamenti articolari, data la sua bassa resistenza all'usura per sfregamento. L'attività di tesi è stata quindi incentrata sulla definizione dei bagni elettrolitici e del ciclo elettrico ottimali per realizzare, con tecnica PEO, rivestimenti in grado di conferire una buona resistenza allo sfregamento alla lega di titanio. Il raggiungimento degli obiettivi prefissati è stato valutato attraverso una caratterizzazione microstrutturale e tribologica del rivestimento.

Antennal courtship and functional morphology of tyloids in the parasitoid wasp Syrphoctonus tarsatorius (Hymenoptera: Ichneumonidae: Diplazontinae)

Courtship behaviour and associated morphological characters are believed to evolve under diversifying sexual selection. In Hymenoptera, sexually dimorphic antennal structures, the 'tyloids', show a large variability. Although crucial for functional interpretation, the link between tyloid morphology and courtship behaviour has gained only limited attention. Here, we investigate antennal morphology and antennal courtship in the parasitoid wasp Syrphoctonus tarsatorius (Hymenoptera: Ichneumonidae: Diplazontinae). We confirm the glandular nature of the tyloids by light and scanning electron microscopy. Moreover, we report a new form of antennation during courtship, antennal double-coiling, which links morphology and behaviour by bringing the tyloids in direct contact with the antennae of the female, thus probably facilitating the transfer of a contact pheromone. We show that a change in haemolymph pressure is the activator of the antennal movement and that it can be reproduced in the laboratory using amputated antennae. Investigations of antennal structure and movement in three additional hymenopteran species suggest that the number and location of tyloids coincide with the modality of antennal coiling. Our method for simulating antennal movement will enable retrieving information about courtship behaviour from museum specimens, thus leading to a better understanding of the evolution of courtship behaviour in Hymenoptera.

Social Cognition And Functional Brain Imaging: Extending The Broader Autism Phenotype

Evidence suggests that the social cognition deficits prevalent in autism spectrum disorders (ASDs) are widely distributed in first degree and extended relatives. This ¿broader autism phenotype¿ (BAP) can be extended into non-clinical populations and show wide distributions of social behaviors such as empathy and social responsiveness ¿ with ASDs exhibiting these behaviors on the lower ends of the distributions. Little evidence has previously shown relationships between self-report measures of social cognition and more objective tasks such as face perception in functional magnetic resonance imaging (fMRI) and event-related potentials (ERPs). In this study, three specific hypotheses were addressed: a) increased social ability, as measured by an increased Empathy Quotient, decreased Social Responsiveness Scale (SRS-A) score, and increased Social Attribution Task score, will predict increased activation of the fusiform gyrus in response to faces as compared to houses; b) these same measures will predict N170 amplitude and latency showing decreased latency and increased amplitude for faces as compared to houses with increased social ability; c) increased amygdala volume will predict increased fusiform gyrus activation when viewing faces as compared to houses. Findings supported all of the hypotheses. Empathy scores significantly predicted both right FFG activation [F(1,20) = 4.811, p = .041, ß = .450, R2 = 0.20] and left FFG activation [F(1,20) = 7.70, p = .012, ß = .537, R2 = 0.29]. Based on ERP results increased right lateralization face-related N170 was significantly predicted by the EQ [F(1,54) = 6.94, p = .011, ß = .338, R2 = 0.11]. Finally, total amygdala volume significantly predicted right [F(1,20) = 7.217, p = .014, ß = .515, R2 = 0.27] and left [F(1,20) = 36.77, p < .001, ß = .805, R2 = 0.65] FFG activation. Consistent with the a priori hypotheses, traits attributed to the BAP can significantly predict neural responses to faces in a non-clinical population. This is consistent with the face processing deficits seen in ASDs. The findings presented here contribute to the extension of the BAP from unaffected relatives of individuals with ASDs to the general population. These findings also give continued evidence in support of a continuous distribution of traits found in psychiatric illnesses in place of a traditional, dichotomous ¿all-or-nothing¿ diagnostic framework of neurodevelopmental and neuropsychiatric disorders.

Examining The Genetic, Social-Cognitive, And Neural Correlates Of Autism Spectrum Disorder Behaviors In Typically Developing Chi

Autism spectrum disorders (ASD) are pervasive developmental disorders that affect approximately 1 in 50 children (Blumberg et al., 2013). Due to the social nature of the deficits that characterize the disorders, many have classified them as disorders of social cognition, which is the process that individuals use in order to successfully interact with members of their own species (Frith & Frith, 2007). Previous research has typically neglected the spectrum nature of ASD in favor of a more categorical approach of ¿autistic¿ versus ¿non-autistic,¿ but the spectrum requires a more continuous approach. Thus, the present study sought to examine the genetic, social-cognitive, and neural correlates of ASD-like traits as well as the relationship between these dimensions in typically developing children. Parents and children completed several quantitative measures examining several areas of social-cognitive functioning, including theory of mind and social functioning, restricted/repetitive behaviors and interests, and adaptive/maladaptive functioning. Children were also asked to undergo an EEG and both parents and children contributed a saliva sample that was used to sequence four single nucleotide polymorphisms (SNPs) of the OXTR gene, rs1042778, rs53576, rs2254298, and rs237897. We successfully demonstrated a significant relationship between behavioral measures of social-cognition and differences in face perception via the N170. However, the directionality of these relationships varied based on the behavioral measure and particular N170 difference scores. We also found support for the associations between the G_G allelic combination of rs1042778 and the A_A and A_G allelic combinations of rs2254298 and increased ASD-like behavior with decreased social-cognitive functioning. In contrast, our results contradict previous findings with rs237897 and imply that individuals with the A_A and A_G genotypes are less similar to those with ASD and have higher social cognitive functioning than those with the G_G genotype. In conclusion, we have demonstrated the existence of ASD-like traits in typically developing children and have shown a link between behavioral, genetic, and neural correlates of social-cognition. These findings demonstrate the importance of considering autism as a spectrum disorder and provide support for the move to a more continuous approach to neurodevelopmental disorders.

Thinner cortex in the frontal lobes in mentally disordered offenders

Antisocial and violent behaviour have been associated with both structural and functional brain abnormalities in the frontal and the temporal lobes. The aim of the present study was to assess cortical thickness in offenders undergoing forensic psychiatric assessments, one group with psychopathy (PSY, n=7) and one group with autism spectrum disorder (ASD, n=7) compared to each other as well as to a reference group consisting of healthy non-criminal subjects (RG, n=12). A second aim was to assess correlation between scores on a psychopathy checklist (PCL-SV) and cortical thickness. Magnetic resonance imaging (MRI) and surface-based cortical segmentation were used to calculate cortical thickness. Analyses used both regions of interest and statistical maps. When the two groups of offenders were compared, there were no differences in cortical thickness, but the PSY group had thinner cortex in the temporal lobes and in the whole right hemisphere compared to RG. There were no differences in cortical thickness between the ASD group and RG. Across subjects there was a negative correlation between PCL-SV scores and cortical thickness in the temporal lobes and the whole right hemisphere. The findings indicate that thinner cortex in the temporal lobes is present in psychopathic offenders and that these regions are important for the expression of psychopathy. However, whether thinner temporal cortex is a cause or a consequence of the antisocial behaviour is still unknown.

Social Cognition, Face Processing, and Oxytocin Receptor Single Nucleotide Polymorphisms in Typically Developing Children

Recent research has provided evidence of a link between behavioral measures of social cognition (SC) and neural and genetic correlates. Differences in face processing and variations in the oxytocin receptor (OXTR) gene have been associated with SC deficits and autism spectrum disorder (ASD) traits. Much work has examined the qualitative differences between those with ASD and typically developing (TD) individuals, but very little has been done to quantify the natural variation in ASD-like traits in the typical population. The present study examines this variation in TD children using a multidimensional perspective involving behavior assessment, neural electroencephalogram (EEG) testing, and OXTR genotyping. Children completed a series of neurocognitive assessments, provided saliva samples for sequencing, and completed a face processing task while connected to an EEG. No clear pattern emerged for EEG covariates or genotypes for individual OXTR single nucleotide polymorphisms (SNPs). However, SNPs rs2254298 and rs53576 consistently interacted such that the AG/GG allele combination of these SNPs was associated with poorer performance on neurocognitive measures. These results suggest that neither SNP in isolation is risk-conferring, but rather that the combination of rs2254298(A/G) and rs53576(G/G) confers a deleterious effect on SC across several neurocognitive measures. Copyright 2014. Published by Elsevier Ltd.