Academic literature on the topic 'Triplet repeat diseases'

Dynamic mutations in some human genes containing trinucleotide repeats are associated with severe neurodegenerative and neuromuscular disorders—known as Trinucleotide (or Triplet) Repeat Expansion Diseases (TREDs)—which arise when the repeat number of triplets expands beyond a critical threshold. While the mechanisms causing the DNA triplet expansion are complex and remain largely unknown, it is now recognized that the expandable repeats lead to the formation of nucleotide configurations with atypical structural characteristics that play a crucial role in TREDs. These nonstandard nucleic acid forms include single-stranded hairpins, Z-DNA, triplex structures, G-quartets and slipped-stranded duplexes. Of these, hairpin structures are the most prolific and are associated with the largest number of TREDs and have therefore been the focus of recent single-molecule FRET experiments and molecular dynamics investigations. Here, we review the structural and dynamical properties of nucleic acid hairpins that have emerged from these studies and the implications for repeat expansion mechanisms. The focus will be on CAG, GAC, CTG and GTC hairpins and their stems, their atomistic structures, their stability, and the important role played by structural interrupts.

Numerous human transcripts contain tandem repeats of trinucleotide motifs, the function of which remains unknown. In this study we used the available gene expression EST data to characterize the abundance of a large group of these transcripts in different tissues and determine the mRNAs which had the highest contribution to the observed levels of transcripts containing different types of the CNG repeats. A more extensive characteristics was performed for transcripts containing the CUG repeats, and those encoding the repeat-binding proteins. The scarcity of double-stranded CUG repeats as well as various proportions of the single-stranded and double-stranded CUG repeat-binding proteins were revealed in the studied transcriptomes. The observed correlated levels of transcripts containing single-stranded CUG repeats and of proteins binding single-stranded CUG repeats may imply that in addition to transcripts which only provide binding sites for these proteins there may be a substantial portion of the transcripts whose metabolism is directly regulated by such proteins. Our results showing a highly variable composition of triplet repeat-containing transcripts and their interacting proteins in different tissues may contribute to a better understanding of the mechanism of RNA-mediated pathogenesis in triplet repeat expansion diseases.

ABSTRACT Despite substantial progress in understanding the mechanism by which expanded CTG/CAG trinucleotide repeats cause neurodegenerative diseases, little is known about the basis for repeat instability itself. By taking advantage of a novel phenomenon, we have developed a selectable assay to detect contractions of CTG/CAG triplets. When inserted into an intron in the APRT gene or the HPRT minigene, long tracts of CTG/CAG repeats (more than about 33 repeat units) are efficiently incorporated into mRNA as a new exon, thereby rendering the encoded protein nonfunctional, whereas short repeat tracts do not affect the phenotype. Therefore, contractions of long repeats can be monitored in large cell populations, by selecting for HPRT+ or APRT+ clones. Using this selectable system, we determined the frequency of spontaneous contractions and showed that treatments with DNA-damaging agents stimulate repeat contractions. The selectable system that we have developed provides a versatile tool for the analysis of CTG/CAG repeat instability in mammalian cells. We also discuss how the effect of long CTG/CAG repeat tracts on splicing may contribute to the progression of polyglutamine diseases.

Objective:To study the role of diabetes, gynecomastia and CAG triplet repeat size as disease modifying factors of neurologic expression in spinal bulbar muscular atrophy (SBMA, Kennedy's disease).Methods:Twenty unrelated SBMApatients with confirmatory genetic testing were reviewed. Patterns of neurologic involvement were assessed (e.g. bulbar, asymmetric, proximal, distal, motor and sensory). Slopes of disease progression were calculated from serial quantified neurologic examinations. Patterns of neurologic involvement and course were correlated to the presence of diabetes, gynecomastia and triplet repeat size.Results:Diabetes or glucose impairment occurred in nine and 11 had gynecomastia. Patterns of neurologic involvement and rates of progression did not correlate with these endocrine diseases or triplet repeat sizes. Correlation was seen between number of CAG repeats and age of onset weakness (r = -0.53, r2 = 29%, p = 0.01).Conclusion:The specific neurotoxic effect of expanded CAGs appears limited to age of onset weakness in SBMA. Although significant, only 29% of the variability in onset age could be accounted for by polyglutamine size suggesting the importance of other unidentified factors. In this series diabetes or glucose impairment was more common than previously reported and, like gynecomastia, did not correlate with size of triplet repeats, severity or patterns of neurologic involvement. Modifying factors other than diabetes, gynecomastia or triplet repeat size are suggested in disease expression.

Trinucleotide repeat expansion disorders (TREDs) are a group of dominantly inherited neurological diseases caused by the expansion of unstable repeats in specific regions of the associated genes. Expansion of CAG repeat tracts in translated regions of the respective genes results in polyglutamine- (polyQ-) rich proteins that form intracellular aggregates that affect numerous cellular activities. Recent evidence suggests the involvement of an RNA toxicity component in polyQ expansion disorders, thus increasing the complexity of the pathogenic processes. Neurodegeneration, accompanied by reactive gliosis and astrocytosis is the common feature of most TREDs, which may suggest involvement of inflammation in pathogenesis. Indeed, a number of immune response markers have been observed in the blood and CNS of patients and mouse models, and the activation of these markers was even observed in the premanifest stage of the disease. Although inflammation is not an initiating factor of TREDs, growing evidence indicates that inflammatory responses involving astrocytes, microglia, and the peripheral immune system may contribute to disease progression. Herein, we review the involvement of the immune system in the pathogenesis of triplet repeat expansion diseases, with particular emphasis on polyglutamine disorders. We also present various therapeutic approaches targeting the dysregulated inflammation pathways in these diseases.

La dystrophie myotonique de type 1 (DM1) est la dystrophie la plus fréquente chez l'adulte avec une prévalence estimée à 1 : 8000 individus. C'est une maladie multi-systémique caractérisée par des atteintes musculaires, cardiaques, cognitives et digestives responsables d'une réduction de l'espérance et de la qualité de vie des patients. Elle est causée par une expansion anormale de répétitions CTG en 3'UTR du gène DMPK. Dans la population générale, le nombre de répétitions est inférieur à 35 CTG tandis qu'il dépasse 50 CTG et peut atteindre jusqu'à plusieurs milliers de répétitions chez les patients. Comme dans d'autres maladies causées par une expansions de triplets répétés, l'expansion CTG est instable chez les patients DM1. La taille des répétitions CTG augmente entre les générations (instabilité intergénérationnelle) et au sein des tissus au cours de la vie des patients (instabilité somatique). Or, le nombre de répétitions héritées ainsi que le niveau d'instabilité somatique corrèlent avec l'âge d'apparition des symptômes ainsi qu'avec leur sévérité. S'attaquer à la mutation elle-même pour stabiliser ou réduire le nombre de répétitions CTG est la voie thérapeutique la plus prometteuse puisque cela permettrait d'agir sur l'ensemble des mécanismes physiopathologiques qui découlent de la présence de la mutation.Dans un premier temps, mes travaux de thèse ont porté sur l'identification de molécules chimiques de repositionnement capables de moduler l'instabilité des répétitions. Le criblage des 1280 molécules de la chimiothèque Prestwick m'a permis d'identifier 39 molécules candidates qui modifient l'expression d'un gène rapporteur suggérant qu'elles pourraient moduler l'instabilité des répétitions. Après étude directe de leur effet sur l'instabilité, j'ai pu exclure quatre de ces molécules qui ne modulent pas l'expression des répétitions. J'ai montré qu'une cinquième molécule, la clomipramine, était capable de moduler l'instabilité des répétitions dans le modèle cellulaire de criblage mais pas dans des modèles cellulaires DM1 murins et humains.Parallèlement, j'ai démontré que RGFP966, un inhibiteur sélectif d'HDAC3, induisait des contractions des répétitions CTG dans des fibroblastes murins DM1 avec environ 650 répétitions CTG. Cet effet semble dépendre de la dose de RGFP966 ou de la taille de la répétition CTG car il n'a pas été reproduit dans des fibroblastes humains DM1 avec 350 CTG. Une approche RNA-seq dans les cellules murines traitées avec RGFP966 a permis d'identifier plusieurs gènes candidats impliqués dans la réplication de l'ADN, comme modificateurs possibles de l'instabilité. J'ai également montré une diminution de la transcription bidirectionnelle de DMPK associée à une probable hyperméthylation en aval des répétitions dans les cellules murines DM1. En conclusion, mes données suggèrent que RGFP966 module l'instabilité des répétitions CTG dans la DM1 par de multiples mécanismes, incluant potentiellement une modification de la structure chromatinienne au locus DM1 et des altérations de la réplication de l'ADN.L'ensemble de mon projet de thèse a permis de progresser dans la compréhension des mécanismes de l'instabilité et dans l'identification de molécules chimiques modulant la dynamique de l'instabilité. Mes travaux de thèse ont également permis de relever les limites de chacun des modèles utilisés et la complexité d'identifier de petites molécules modifiant la dynamique des triplets CTG en utilisant des modèles cellulaires rapporteurs. Par ailleurs, j'ai participé au développement pour la DM1 du séquençage à longues lectures (avec et sans amplification), un nouvel outil rapide et très informatif pour analyser la mosaïque somatique
Myotonic dystrophy type 1 (DM1) is the most common dystrophy in adults, with an estimated prevalence of 1:8000 individuals. It is a multisystemic disease characterized by muscle, cardiac, cognitive, and digestive impairments, which contribute to a reduction in both life expectancy and quality of life for patients. DM1 is caused by an abnormal expansion of CTG repeats in the 3'UTR of the DMPK gene. In the general population, the number of repeats is under 35 CTG, whereas in patients, it exceeds 50 CTG and can reach several thousand repeats. As in other diseases caused by repeat expansions, the CTG expansion in DM1 is unstable. The repeat size increases across generations (intergenerational instability) and within tissues during a patient's lifetime (somatic instability). The number of inherited repeats and the level of somatic instability correlate with the age of onset and severity of symptoms. Thus, targeting the mutation itself to stabilize or reduce CTG repeat length is the most promising therapeutic strategy, as it would address all the pathophysiological mechanisms resulting from the mutation.Initially, my thesis work focused on identifying repositioned chemical molecules capable of modulating repeat instability. Screening the 1280 molecules from the Prestwick Chemical Library allowed me to identify 39 candidate molecules that alter the expression of a reporter gene, suggesting they could modulate repeat instability. After directly studying their effect on instability, I excluded four of these molecules that do not modulate repeat expression. I demonstrated that a fifth molecule, clomipramine, can modulate repeat instability in the screening cell model but not in murine and human DM1 fibroblasts.Concurrently, I showed that RGFP966, a selective HDAC3 inhibitor, induced contractions of CTG repeats in murine DM1 fibroblasts with approximately 650 repeats. This effect appears to depend on the dose of RGFP966 or the size of the CTG repeat, as it was not replicated in human DM1 fibroblasts with 350 CTG repeats. An RNA-seq approach in murine cells treated with RGFP966 identified several candidate genes involved in DNA replication as possible modifiers of instability. I also showed a decrease in bidirectional DMPK transcription associated with a probable hypermethylation downstream of the repeats in murine DM1 cells. In conclusion, my data suggest that RGFP966 modulates CTG repeat instability in DM1 through multiple mechanisms, potentially including chromatin structure modification at the DM1 locus and alterations in DNA replication.Overall, my thesis project contributed to the understanding of repeat instability mechanisms and the identification of chemical compounds that modulate instability dynamics. My work also highlighted the limitations of each model used and the complexity of identifying small molecules that alter CTG triplet dynamics in reporter cell models. Additionally, I participated in developing long-read sequencing (with and without amplification) for DM1, providing a rapid and highly informative new tool for the analysis of somatic mosaicism

A PCR strategy showed that a number of total mtDNA molecules was significantly decreased (~30%) in the striatum (no reduction in the cortex and cerebellum) of 24-month old HD mice, but not a 15 months of age, when compared to wild-type mice, suggesting mtDNA depletion is a progressive rather than a developmental phenomenon. In light of the ~30% reduction of total mtDNA in the striatum, expression levels of the mitochondrial DNA-encoded respiratory complex enzymes, cytochrome b(Cytb), cytochrome c oxidase I (COI) and cytochrome c oxidase II (COII) were investigated in different brain regions of HD mice. At ~25 months of age, there were no significant differences in mRNA levels of CoII and Cytb in any brain region (striatum, cortex and cerebellum) studied when compared to normal littermates. However, HD mice showed significantly decreased CO-I protein levels and marginally decreased CoI mRNA levels in the striatum. Reduced levels of mtDNA may be caused by decreased replication of mtDNA or increased oxidative damage of mtDNA. Increased levels of 8-OHdG, a marker of increased oxidative stress, were detected in the dorsomedial, dorsolateral and ventromedial striatum, but not in the cortex of 24-month old HD mice providing direct evidence that increased oxidative stress specifically occurs in the striatum of HD mice. As no alterations in the mitochondrial transcription factor (mtTFA) in the striatum of HD mice could be detected, it is likely that mtDNA depletion in the HD mice is caused by increased levels of oxidative stress rather than decreased replication. The results provide a basis for further studies investigating how mutant huntingtin causes increased levels of oxidative stress and for identifying novel therapeutic targets.

DNA replication is essential to allow faithful inheritance of the genome. In mammalian cells, many replication origins are grouped within 200-400 kb regions called replication clusters, which are in turn enclosed in large replication domains (Méchali 2010; Cayrou et al. 2011). This hierarchical organisation is required for the temporal and spatial control of DNA replication and it allows modulating origin activation locally within clusters and globally at the level of replication domains (Yekezare et al. 2013). During G1 several initiation sites are licensed, but only a subset is activated in the following S-phase (Ge et al. 2007; Blow & Ge 2009; Méchali 2010). Furthermore, origins fire stochastically within clusters and in different positions among cells in a population (Hyrien et al. 2003; Gilbert 2007; Méchali 2010). Remarkably, origin redundancy and plasticity are intrinsic features of the mammalian replication process necessary to face changes in chromatin organisation occurring during development and cell differentiation and to overcome replication stress (Cortez 2015; Alver et al. 2014; Palumbo et al. 2013; Zeman & Cimprich 2014). The temporal regulation of origin firing is established at the level of replication domains, which are conserved among subsequent cell cycles and classified as early, mid or late replicating according to their timing of activation throughout the S-phase (Hiratani et al. 2008; Pope & Gilbert 2013; Rivera-Mulia & Gilbert 2016b). Replication profiles define the replication program in each cell type and are modulated according to the diverse developmental and differentiation cellular stages, as well as in response to replication stress (Palumbo et al. 2013; Courbet et al. 2008; Anglana et al. 2003). Replication profiles are determined by several parameters, including fork rates, origin choice and alternative replication patterns (i.e. unidirectional and asynchronous forks and pause/arrest events), and their regulation is implied by the flexible nature of the mammalian replication process. Hence, not only origin position but the whole replication dynamics must be taken into consideration to better elucidate the complex phenomena associated with mammalian DNA replication (Prioleau & MacAlpine 2016; Hyrien 2015; Palumbo et al. 2013). In this frame, single-molecule techniques represent the most appropriate tool to detect the intrinsic plasticity and cell-to-cell variability of mammalian genomes (Tuduri et al. 2010; Técher et al. 2016; Prioleau & MacAlpine 2016). Here, I evaluated how replication profiles are regulated in response to endogenous and exogenous replication stress conditions. In particular, I considered the modulation of the mammalian replication program in relation to sequence organisation, cell growth and differentiation. For this purpose, different cellular models were used. First, I focused on the effects of a trinucleotide repeat (TNR) expansion on DNA replication. TNR are among the most unstable genomic regions and variations of their length are implicated in many human neurodegenerative disorders (McMurray 2010; Lee & McMurray 2014). Expanded repeats are prone to form stable unusual DNA secondary structures (Mirkin & Mirkin 2007; Krasilnikova & Mirkin 2004), which are a well-known source of replication stress (Zeman & Cimprich 2014; Magdalou et al. 2014; León-Ortiz et al. 2014). A replication-based mechanism is largely accepted to be at the origin of expansion and instability of several TNR, including also the GAA-repeat expansion responsible for Friedreich’s ataxia (FRDA). To better understand this mechanism, different experimental systems from yeast to transfected or engineered human cells were employed (Follonier et al. 2013; Chandok et al. 2012; Kim et al. 2008), but none of them displayed the huge amount of repeats observed in patients’ cells. Thus, to understand how replication profiles are modulated in the presence of long repetitive sequences in the endogenous context, I used human lymphoblastoid cell lines derived from FRDA patients carrying an homozygous GAA-repeat expansion within intron 1 of the Frataxin (FXN) gene. In the presence of the GAA-repeat expansion an alteration of the replication timing was observed by interphase FISH and wide changes in the replication profiles were demonstrated. Indeed, in mutant cells, according to the frequency of duplicated FISH spots, the replication of the FXN gene was slowed or delayed during the first half of the S-phase. This effect was normalised in the latter part when both normal and expanded alleles complete their replication. Further, by molecular combing replication dynamics was monitored in a large genomic region harbouring FXN. By this approach I verified that the most relevant effect associated with the presence of the GAA-repeat expansion was the recruitment of additional dormant origins firing downstream the repeat, which can be considered as a rescue mechanism to assure replication of mutant alleles. As a consequence of dormant origin activation, a switch of the prevalent direction by which the expanded GAA-repeat is replicated was observed, indicating that the origin-switch model for TNR instability conform to the case of the GAA-repeat expansion, similarly to what observed at the FMR1 locus (Gerhardt et al. 2014). Same conclusions were reached in parallel in another study (Gerhardt et al. 2016). Remarkably, a strong reduction of unidirectional fork length was observed in mutant alleles with respect to the normal sequence and this may be considered as a second effect of the GAA-repeat expansion. In line with results obtained at the FMR1 locus (Gerhardt et al. 2014), in the present study recurrent paused/arrested forks were recorded in proximity of the short GAA-repeat in one of the normal cell lines analysed, suggesting a possible impact of the non-pathological GAA-repeat on fork progression. Interestingly, it was recently demonstrated an high occurrence of fork pausing at the expanded GAA-repeat in both undifferentiated and differentiated FRDA cells and these events were hypothesised to be caused by a collision between the replication and transcription machineries (Gerhardt et al. 2016). Understanding whether the origin-switch is the cause or instead a consequence of the GAA-repeat expansion will be necessary and future investigations will primarily contribute to clarify this aspect. Secondly, I evaluated the effects of increased and unbalanced dNTP pools on the replication program. To ensure completion of DNA replication and to avoid genome instability, the balance among dNTP supply, degradation and consumption must be tightly controlled according to the rate of DNA synthesis (Rampazzo et al. 2010; Chabes & Stillman 2007; Chabosseau et al. 2011; Bester et al. 2011). Indeed, the limiting availability of nucleotide precursors has been widely demonstrated to induce replication stress by slowing fork progression and leading to activation of the DNA damage response (Anglana et al. 2003; Courbet et al. 2008). Conversely, the consequences of an increased supply of dNTPs were only partially depicted in yeast mutants, where replication forks move faster upon ribonucleotide reductase overexpression (Poli et al. 2012). Instead, the effects in mammalian cells are still unknown. Thus, here I used human primary fibroblasts and THP1 monocytes with increased and unbalanced dNTP pools due to depletion of SAMHD1, a protein involved in nucleotide metabolism (Franzolin et al. 2013; Miazzi et al. 2014). By this experimental models the replication profiles were analysed genome-wide by molecular combing in comparison to normal cells. My results indicated that independently of the dNTP pool imbalances, DNA replication proceeds mostly undisturbed in mutated and control fibroblasts, both in physiological and perturbed growth conditions. In contrast, an unexpected slow down of replication forks and a consequent increase in origin firing were detected in SAMHD1-depleted THP1 cells with respect to the wildtype cell line. This differential response to the high availability of nucleotide precursors may be ascribed either to a supposed cell type-specific activity of SAMHD1 or to the development of an adaptive phenotype in mutant fibroblasts compensating for the SAMHD1 depletion. Future goals will be to confirm these hypotheses and to evaluate the effects associated with fork stalling and restart. Finally, I considered how replication dynamics are modulated during cell differentiation. It is well known that replication timing and replication profiles are cell type-specific and regulated according to changes in chromatin organisation occurring during development and cell differentiation (Palumbo et al. 2013; Hiratani et al. 2010; Hiratani et al. 2008). Hence, I assessed whether DNA replication is affected upon forced cell cycle reactivation. In particular, terminally differentiated mouse myotubes forced to re-enter the cell cycle were analysed by molecular combing in comparison to proliferating myoblasts. A significant reduction of fork rates was detected in reactivated myotubes with respect to proliferating muscle cells, resembling the effects seen under replication stress. This result is in line with previous evidence indicating that myotubes fail to complete their replication, as they are not able to properly expand their dNTP pools (Pajalunga et al. 2010; Pajalunga et al. 2017). Accordingly, after addition of nucleotide precursors the reduction of fork rate was partially ameliorated. However, the number of activated origins in each replication cluster was comparable between myotubes dosed with deoxynucleosides and myoblasts, suggesting that some regions along the genome are left under-replicated. Thus, the replication failure detected upon forced cell cycle re-entry may be ascribed not only to the depletion of nucleotide precursors, but also to the inability of these cells to recruit additional origins compensating for the reduction of fork speed. Interestingly, I found an increased proportion of unidirectional forks in reactivated myotubes when compared to myoblasts, in line with previous data obtained in human primary fibroblasts (Palumbo et al. 2013). Thus, unidirectional forks could be viewed as a remnant of the modality by which some replication domains are replicated when cells move toward terminal differentiation. In this perspective, understanding whether replication timing may be implicated in the replication impairment observed upon forced cell cycle reactivation remains an intriguing issue to be further unravelled. The complexity of mammalian DNA replication may be ascribed to the intrinsic plasticity of the process, and drawing general conclusions from studies based on individual cellular models may be incautious. The results obtained in this study add new evidence for interpret this complexity, and offer insights for future investigations.
La replicazione del DNA è essenziale per consentire l’accurata trasmissione del materiale genetico durante le successive divisioni cellulari. Nelle cellule di mammifero i siti di inizio sono raggruppati in regioni genomiche di 200-400 kb, definite cluster replicativi, che sono a loro volta racchiusi in più ampi domini di replicazione (Méchali 2010; Cayrou et al. 2011). Questa organizzazione gerarchica consente di controllare il processo replicativo nello spazio e nel tempo tramite la regolazione dell’attivazione delle origini sia a livello locale all’interno dei cluster che a livello globale nei domini di replicazione (Yekezare et al. 2013). Durante la fase G1 del ciclo cellulare le origini vengono “licenziate”, ma solo una parte di esse viene poi attivata nella successiva fase S (Ge et al. 2007; Blow & Ge 2009; Méchali 2010). Inoltre, le origini si attivano in modo stocastico all’interno dei cluster e sono diversamente distribuite nel genoma tra le diverse cellule (Hyrien et al. 2003; Gilbert 2007; Méchali 2010). Pertanto, le origini di replicazione nei mammiferi sono ridondanti e flessibili (Hyrien et al. 2003; Méchali 2010), caratteristiche indispensabili per fronteggiare non solo condizioni di stress replicativo, ma anche cambiamenti dell’organizzazione cromatinica quali quelli che avvengono durante lo sviluppo e il differenziamento cellulare (Cortez 2015; Alver et al. 2014; Palumbo et al. 2013; Zeman & Cimprich 2014). La regolazione temporale dell’attivazione delle origini è stabilita a livello dei domini replicativi, conservati in successivi cicli cellulari e classificati come regioni a replicazione precoce, intermedia e tardiva in base al loro timing di attivazione durante la fase S (Hiratani et al. 2008; Pope & Gilbert 2013; Rivera-Mulia & Gilbert 2016b). I profili replicativi determinano il programma di replicazione specifico di ogni tipo cellulare e sono regolati durante le diverse fasi dello sviluppo e del differenziamento, così come in risposta a stress (Palumbo et al. 2013; Courbet et al. 2008; Anglana et al. 2003). I profili di replicazione sono definiti da vari parametri: oltre alla posizione e alla scelta delle origini, anche la velocità di progressione delle forche e i diversi pattern replicativi alternativi, quali forche unidirezionali, asincrone e in pausa, sono finemente regolati. Per la corretta interpretazione dei complessi fenomeni associati alla replicazione del DNA tutti questi pattern devono essere attentamente considerati (Prioleau & MacAlpine 2016; Hyrien 2015; Palumbo et al. 2013). Pertanto, per riuscire ad apprezzare la plasticità e la variabilità intrinseche del processo replicativo, le tecniche di analisi su singola molecola risultano essere particolarmente appropriate (Tuduri et al. 2010; Técher et al. 2016; Prioleau & MacAlpine 2016). In questo lavoro ho valutato come vengono modulati i profili di replicazione in presenza di stress replicativi endogeni ed esogeni. In particolare, utilizzando diversi modelli cellulari ho analizzato come venga regolata la replicazione del DNA in regioni genomiche instabili, in particolari condizioni di crescita e durante il differenziamento. In primo luogo, ho determinato gli effetti associati alla presenza di sequenze trinucleotidiche ripetute. Le triplette ripetute sono tra le regione del genoma più instabili e variazioni della loro lunghezza sono coinvolte in diverse malattie neurodegenerative (McMurray 2010; Lee & McMurray 2014). La loro caratteristica principale, strettamente collegata alla loro instabilità, è la capacità di formare strutture secondarie insolite (Mirkin & Mirkin 2007; Krasilnikova & Mirkin 2004), che inducono stress replicativo impedendo la normale progressione delle forche (Zeman & Cimprich 2014; Magdalou et al. 2014; León-Ortiz et al. 2014). Si pensa che tra i vari processi metabolici associati al DNA la replicazione abbia un ruolo fondamentale nel promuovere l’espansione e l’instabilità delle triplette ripetute, tra cui anche la ripetizione GAA associata all’atassia di Friedreich (Pearson et al. 2005; Cleary & Pearson 2005). Per capire meglio il meccanismo che sta alla base della loro instabilità sono stati utilizzati diversi modelli sperimentali, dal lievito a cellule umane ingegnerizzate (Follonier et al. 2013; Chandok et al. 2012; Kim et al. 2008). Tuttavia, rispetto ai lunghi tratti ripetuti osservati in cellule di pazienti, il limitato numero di triplette presente in questi modelli non consente uno studio approfondito degli effetti sulla sintesi del DNA. Per questo motivo, ho analizzato i profili replicativi in cellule linfoblastoidi umane derivate da pazienti affetti da atassia di Friedreich e che presentano in omozigosi un’espansione della tripletta GAA nel primo introne del gene Fratassina. In presenza dell’espansione ho osservato alterazioni del timing di replicazione del gene tramite la tecnica della FISH su nuclei in interfase, identificando vari cambiamenti nei profili replicativi della regione analizzata. Nello specifico, in base alla proporzione dei segnali di ibridazione duplicati ho potuto definire un ritardo nella replicazione del gene mutato durante la prima metà della fase S. Questo ritardo viene però recuperato nella parte finale della fase S, in quanto la replicazione del gene viene completata sia negli alleli normali che in quelli con tripletta espansa. Grazie alla tecnica del molecular combing ho poi monitorato le dinamiche di replicazione di una regione genomica di 850 kb contenente il gene Fratassina. Dalle mie analisi è emerso che l’effetto principale associato alla presenza della mutazione è l’attivazione di origini dormienti aggiuntive, situate a valle della tripletta ripetuta e all'interno del gene. Esse possono essere considerate come un meccanismo di recupero per assicurare il completamento della replicazione negli alleli mutati. In conseguenza dell’attivazione di queste origini dormienti, la direzione con cui la tripletta espansa viene replicata cambia, dimostrando che il modello di origin-switch, proposto per descrivere i meccanismi di instabilità delle triplette ripetute, è conforme al caso dell’espansione GAA nel gene Fratassina, come precedentemente osservato anche nel locus FMR1 (Gerhardt et al. 2014). In maniera indipendente un altro gruppo di ricerca è giunto alle mie stesse conclusioni (Gerhardt et al. 2016). Nel corso di queste analisi è emersa una significativa diminuzione della lunghezza delle forche unidirezionali negli alleli espansi, che può essere considerata come un secondo effetto associato alla mutazione nel gene Fratassina. Similmente a quanto osservato nel locus FMR1 (Gerhardt et al. 2014), in questo lavoro sono stati identificati eventi di arresto/pausa delle forche in corrispondenza della corta ripetizione GAA in una delle due linee di controllo analizzate, osservazione che suggerisce un possibile ruolo del tratto ripetuto non patologico sulla progressione delle forche. Recentemente è stato dimostrato che la sequenza GAA espansa è un sito di blocco delle forche replicative, ed è stato ipotizzato che gli eventi di arresto siano dovuti alla collisione tra i complessi proteici coinvolti nella replicazione e nella trascrizione del DNA (Gerhardt et al. 2016). Indagini future devono mirare a capire se il meccanismo di origin-switch osservato in presenza dell’espansione GAA sia la causa dell’espansione stessa o ne sia una conseguenza. Un secondo aspetto che ho considerato riguarda la modulazione del programma replicativo in seguito a innalzamento e sbilanciamento dei pool dei nucleotidi. Sintesi, degradazione e consumo dei precursori del DNA devono essere accuratamente controllati per assicurare la replicazione completa del genoma e per evitare instabilità (Rampazzo et al. 2010; Chabes & Stillman 2007; Chabosseau et al. 2011; Bester et al. 2011). In presenza di quantità limitate di nucleotidi le forche replicative rallentano drasticamente e inducono l’attivazione della risposta al danno del DNA, sottoponendo le cellule a stress replicativo (Anglana et al. 2003; Courbet et al. 2008). Al contrario, le conseguenze di un elevato supporto di nucleotidi sulle dinamiche di replicazione sono state osservate solo in lievito, dove in seguito a sovraespressione dell’enzima ribonucleotide reduttasi è stato dimostrato un aumento della velocità di progressione delle forche (Poli et al. 2012). Gli effetti in cellule di mammifero sono invece ancora sconosciuti. Per questo motivo ho utilizzato come modelli sperimentali fibroblasti primari umani e monociti della linea THP1 con pool nucleotidici alti e sbilanciati a causa della mancanza della proteina SAMHD1 (Franzolin et al. 2013; Miazzi et al. 2014). Con la tecnica del molecular combing mi è stato possibile studiare i profili di replicazione a livello di intero genoma. Ho dimostrato che lo sbilanciamento dei pool dei nucleotidi non influisce sulle dinamiche di replicazione dei fibroblasti primari, né in condizioni fisiologiche né sotto stress replicativo. Al contrario, i monociti THP1 privi di SAMHD1 mostrano un inatteso rallentamento delle forche replicative con conseguente aumento delle origini attivate in ogni cluster. La diversa risposta cellulare osservata in seguito allo sbilanciamento dei pool può essere spiegata da un ruolo specifico di SAMHD1 in funzione del tipo cellulare. In alternativa, è possibile che i fibroblasti abbiano sviluppato un fenotipo adattativo per compensare la mancanza della proteina. Analisi future avranno come scopo quello di verificare la validità di queste due ipotesi e di valutare possibili effetti associati a blocco e ripartenza delle forche replicative. Infine, ho valutato come cambiano i profili di replicazione durante il differenziamento. È noto che timing e dinamiche di replicazione sono specifici del tipo cellulare e sono regolati in base a cambiamenti dell’organizzazione cromatinica che avvengono durante lo sviluppo e il differenziamento (Palumbo et al. 2013; Hiratani et al. 2010; Hiratani et al. 2008). Quindi, ho valutato se la riattivazione forzata del ciclo cellulare in cellule terminalmente differenziate influenzi la replicazione del DNA. Come modello sperimentale ho usato miotubi di topo forzati a rientrare nel ciclo cellulare e li ho analizzati con molecular combing rispetto a mioblasti proliferanti. In seguito a riattivazione forzata, ho osservato una significativa riduzione della velocità delle forche replicative, similmente a quanto visto in condizioni di stress replicativo (Anglana et al. 2003; Palumbo et al. 2010). Questo risultato è in accordo con il fallimento del processo replicativo osservato nei miotubi riattivati e si pensa sia dovuto a un’incapacità di queste cellule di espandere in modo corretto i pool dei nucleotidi (Pajalunga et al. 2010; Pajalunga et al. 2017). In effetti, dopo l’aggiunta dei precursori del DNA nei miotubi riattivati si osserva un parziale miglioramento della velocità delle forche. Nonostante ciò però, il numero di origini attivate in ogni cluster è simile tra miotubi e mioblasti, perciò è ragionevole pensare che regioni del genoma restino non replicate. In conclusione, l’incapacità di completare la replicazione del DNA osservata nei miotubi dopo riattivazione forzata del ciclo cellulare non solo è dovuta alla limitata disponibilità di nucleotidi, ma anche a un difetto nel reclutare origini di replicazione aggiuntive per compensare la riduzione delle velocità. Un secondo aspetto interessante è l'aumentata proporzione di forche unidirezionali nei miotubi riattivati rispetto ai mioblasti, simile a quanto precedentemente osservato in fibroblasti primari umani (Palumbo et al. 2013). In questo caso, le forche unidirezionali visualizzate possono essere considerate come un residuo della modalità con cui alcuni domini replicano quando le cellule vanno verso il differenziamento terminale. Resta interessante capire se anche il timing della replicazione sia alterato dopo riattivazione forzata del ciclo cellulare. La replicazione del DNA è un processo molto complesso caratterizzato da una grande plasticità, pertanto non è consigliato trarre conclusioni generali da studi basati su singoli tipi cellulari. I risultati ottenuti in questo lavoro contribuiscono a interpretare la grande complessità del processo replicativo, offrendo anche nuove prospettive per approfondimenti futuri.

Dissertação de Mestrado, Ciências Biomédicas, 3 de Fevereiro de 2016, Universidade dos Açores.
[...]. Este estudo teve como objetivo o desenvolvimento e a validação de um protocolo de TPPCR para a DMJ. Sessenta e seis amostras de sangue previamente genotipadas por PCR foram utilizadas na otimização e validação da TP-PCR. A robustez da técnica foi testada em 14 amostras de esfregaço bucal de doentes heterozigóticos com um alelo expandido. Os resultados da TP-PCR foram concordantes com os obtidos pela PCR em 65 amostras de sangue. Os resultados obtidos para as amostras de esfregaço bucal foram 100% concordantes com os das amostras de sangue, demostrando que a técnica pode ser aplicada em diferentes tecidos e para uma baixa quantidade de DNA. A TP-PCR desenvolvida e validada para a DMJ deverá constituir uma técnica complementar fiável em laboratórios de diagnóstico, de modo a ultrapassar as limitações da PCR, relacionadas com a falha de amplificação dos alelos expandidos.
ABSTRACT: [...]. The purpose of this study was to develop and validate a TP-PCR protocol for MJD. Sixtysix blood samples previously genotyped by PCR were used to optimize and validate a TP-PCR assay. The robustness of the technique was evaluated using 14 buccal swab samples of heterozygous patients carrying an expanded allele. TP-PCR results were concordant with the ones obtained by PCR for 65 blood samples. The results obtained for the buccal swabs samples were fully concordant with the ones obtained for blood samples showing that the technique can be applied to different tissues and with low amounts of DNA. The TP-PCR developed and validated for MJD should constitute a reliable complementary technique in diagnostic laboratories to overcome the limitations of standard PCR related with the failure in amplification of the expanded alleles.

Introduction: Occult primary breast cancer is very rare, accounting for less than 1% of all cases of breast cancer, generally associated with a poor prognosis. It is defined as a clinically recognizable metastatic carcinoma derived from an undetectable primary breast tumor, with metastasis to the axillary and cervical lymph nodes. Clinical and radiological examinations represent the first steps in the diagnosis, followed by a histological and immunohistochemistry (IHC) analysis, as well as a multidisciplinary team evaluation and therapy - essential for diagnosis and treatment. The most common phenotype is a positive hormone-receptor adenocarcinoma for which there is no clear consensus about optimal management, however a standard approach is axillary lymph node (ALN) dissection. Ipsilateral mastectomy, neoadjuvant chemotherapy and radiotherapy are controversial but may be acceptable in selected cases. Case report: A 72-year-old woman with a history of colon adenocarcinoma surgically treated, presented with an axillary mass of rapid growth. Uponn physical examination, a 5 cm mass in the left axilla and a palpable ipsilateral supraclavicular lymph node (SCLN) were identified, without any evidence of a breast lesion. imaging analysis with bilateral mammography, ultrasonography and breast magnetic ressonance imaging showed suspicious axillary and SC adenopathy, both on the left side; no abnormal breast findings. She was submitted to core biopsy and IHC analysis, andan invasive triple negative metastatic breast cancer was diagnosed. The patient underwent neoadjuvant chemotherapy with cyclophosphamide / doxorubicin, evolving with disease progression, so the regimen was modified to carboplatin. There was no response to treatment, with persistent growing of the lesion. Neoadjuvant chemotherapy was interrupted and surgery was performed to resect the ALN and the SCLN. It was a difficult surgery due to the extension of the axillary mass, in conjunction with adherence to the subclavian vein. A histologic analysis confirmed the inicial diagnosis of metastatic breast cancer. Surgery was followed by radiotherapy, but disease progression was fast. She manifested a large axillary recurrence and progressed to death 4 months after the beginning of treatment. This case report describes how challeging occult breast cancer can be, specially when associated with an unusual presentation such as a triple negative phenotype and SC adenopathy. At first, the hypothesis of colon metastasis was proposed due to the poor reponse to chemotherapy. Despite being submited to the standard approach proposed and supported by literature, the aggressive and rapid progression to death represents an obvious need to discuss other treatment options for occult breast carcinoma with unusual presentations, such as negative hormone-receptor.

Background: Stage IV triple-negative breast cancer has a high mortality rate, and the treatment strategy will be based on the presence of biomarkers, disease burden, need for a response rate, and treatment tolerability. Among the various management modalities and effective treatments, capecitabine is a frequently used option due to its known benefits and relatively good tolerance. However, there are several commonly known adverse effects when using capecitabine, including non-immune hemolytic anemia, a very rare and unexpected side effect. Capecitabine is a form of fluoropyrimidine that is hypothesized to affect the structure of the red blood cell membrane, resulting in the destruction of these cells. Case report: A 71-year-old woman with stage IV triple-negative breast cancer with bone and skin metastases, negative PDL1, and germline mutations in BRCA1/2, with progression disease at first-line chemotherapy. Capecitabine was started at a dose of 2,000 mg/m2 /day, and after two cycles, she developed cytopenia, in addition to increased bilirubin and LDH, leading to the suspicion of hemolysis. She was evaluated by the hematological medical team with complementary tests such as reticulocytes, haptoglobin, and coombs D, the latter negative. Medullary infiltration was ruled out. Capecitabine has been suspended for 15 days, with normalization of tests. When it was reintroduced, there was a new alteration in laboratory tests. In the end, it was decided to permanently discontinue the drug, despite the clinical and radiological response. Final Comments: We present a very complex and challenging clinical case of a patient with metastatic breast cancer undergoing palliative treatment. Although the patient’s disease was controlled with the use of capecitabine, cytopenia developed with suspected medication-induced non-immune hemolytic anemia. This shows the usual complexity of treating patients with drugs that have both acute and chronic side effects.

Introduction: Breast cancer is a heterogeneous disease, divided into many subtypes, the most common being invasive ductal. Breast tumors can be derived from epithelial tissue or mesenchymal cells. Most malignant breast tumors are made up of a single type of tumor. Cases where there are two or more types of malignant tumors occurring at the same time are rare. We report a case with different types of malignancies, i.e., non-special invasive ductal carcinoma, metaplastic carcinoma, and squamous cell carcinoma occurred together. Case report: A woman, 56 years old, with no family history of gynecological neoplasia, sought care referring to a painful and erythematous nodule in her left breast for about 25 days. On physical examination, a 15×11 cm, slightly mobile nodule with irregular borders was noticed in the left breast and palpable axillary lymph nodes. The mammogram showed an isodense nodule with internal calcifications, measuring 10.2×9.4×7.2 cm, in the superolateral quadrant (QSL) of the left breast (ME) — BI-RADS V. The central biopsy showed nonspecial invasive breast carcinoma (NOS), triple-negative, and KI67 30%. She underwent neoadjuvant chemotherapy, and 5 months after the start, she showed evidence of tumor progression. The tumor was ulcerated and occupying the entire QSL of ME and on palpation of the armpit, multiple hardened lymph nodes on the left. Left radical mastectomy with axillary lymphadenectomy was performed. The anatomopathological examination of the specific specimen revealed different tumor types: ulcerated and moderately differentiated squamous cell carcinoma, invasive metaplastic breast carcinoma, with a negative immunohistochemical profile for RP, ER, and HER2, positive cytokeratin 7, and 70% Ki67. Two months after the mastectomy, she had tumor recurrence and underwent a new surgical approach. Plastron histopathology showed squamous cell carcinoma infiltrating skin, negative estrogen and progesterone receptors, and positive p63. She underwent radiotherapy and is currently being followed up, with no signs of recurrence.

A 62 year old man with vWD has suffered from repeated episodes of melaena - his son and daughter have inherited the disorder with few symptoms so far. Laboratory findings in them include consistently prolonged bleeding times, normal factor VIII coagulant activity and decreased ristocetin cofactor activity. Levels of von Willebrand factor (vWf) antigen were lower measured by immunoradiometric assay than by Laurell immunoelectrophoresis. Analysis of vWf structure by SDS agarose gel electrophoresis showed loss of only the large multimers in plasma and the triplet structure of the smaller multimers showed sub-bands more intense than normal. However, the platelets contain the whole series of multimers with a similar pattern to normal. This was suggestive of type IIB vWD but agglutination of the patients' platelet-rich plasma with low concentrations of ristocetin was not enhanced. Agglutination was reduced compared to normal platelet-rich plasma at a final ristocetin concentration of 1 and 2 mg/ml and absent at a final concentration of 0.5 mg/ml. Platelet-type vWD was eliminated as addition of normal plasma or cryoprecipitate to patient's platelet-rich plasma did not produce spontaneous aggregation.The patient has never had thrombocytopenia. We feel that this family demonstrates further the heterogeneity of vWD.

Introduction: The triple-negative breast cancer (TNBC) is one of the most aggressive types of breast cancer, corresponding to about 15% to 20% of invasive breast tumors. They are those tumors that in immunohistochemistry do not express homone receptors and epidermal growth factor type 2 (cerbB2). This tumor phenotype does not yield many treatment options, beyond standard chemotherapy, and within this context, the evidence of some markers of this type of tumor may contribute to the discovery of more effective types of treatment. Case report and Objectives: The aim of this study was to define predictive and prognostic factors in TNBC that could be related with a pathologic complete response after neoadjuvant chemotherapy treatment. Methods: A descriptive and retrospective study, a case series type, in women with TNBC who had underwent neoadjuvant chemotherapy and surgery at the Mastology Service of Maternidade Escola Assis Chateaubriand – Brazil - from May 2015 to June 2020. A statistical analysis was performed considering the 5% significance level. Results: From 108 women, only 47 were included in the study, with median age of 49 years (+14 years); about 30 (42.6%) had a family history of breast cancer in first or seconddegree relatives. About 44 (93.6%) cases were classified as invasive ductal tumor and grade II or III; the value of Ki67 greater than 14% was evidenced in 46 (97.9%) women and 26 (55.3%) had clinical stage III. Pathologic complete response to chemotherapy was evidenced in 16 (34%) cases, partial response in 13 (27.7%) and no response in 18 (38.3%) cases. The latter cases correspondeded to those who had stable or progression of disease. There was recurrence in 13 (27.7%) women, about 8 distant metastases, with the lungs as the most frequent site of metastasis followed by the brain. Eleven patients, about 23.4%, died. In the survival analysis of the studied population, the overall survival was 5.6 months and disease-free survival was 19.4 months. No association was observed in the study between the outcome of pathologic complete response to neoadjuvant chemotherapy and anatomopathological characteristics of the tumor. Conclusion: The results of this study did not show statistical significance to determine the possible predictive and prognostic factors for obtaining a complete clinical response to TNBC in a public reference service for the treatment of breast cancer, where there is no genetic signature, PDL1 status or access to differentiated treatment for such a heterogeneous tumor profil. This shows a need for further studies in order to understand this disease and for greater accessibility to high-cost exams and more effective treatments.

Introduction: Breast cancer is one of the neoplasms that most cause death in women. Among these, there are some subtypes of greater biological aggressiveness, such as triple negative and HER overexpressed, which are associated with greater recurrence and mortality. Adenoid cystic carcinoma (ACC), salivary gland type, represents less than 0.1% of primary breast carcinomas and has indolent biological behavior and favorable prognosis compared with nonspecial triplenegative types. Case Report: A 51-year-old woman diagnosed with locally advanced ACC in the right breast, with negative immunohistochemistry for hormone receptors and HER2, underwent quadrantectomy with upfront axillary dissection, followed by adjuvant radiotherapy. After 12 years of diagnosis, she presented significant back pain, with magnetic resonance imaging scan evidencing bone lesion without medullary involvement in D7 and L1 suggestive of the secondary implant. Anatomopathology revealed the same histology as the primary tumor. Re-evaluation of chest tomography showed progression of pulmonary disease, 5 months after diagnosis of the first metastasis, underwent segmentectomy, with descriptive pathology identical to the initial lesion. Due to the oligoprogression and tumor type, somatic genetic research of the lung material was requested, which revealed a mutation in the NTRK gene, patient is still waiting for Larotrectinib in court. Discussion: The tumor has an unusual histological type, rare occurrence, slow progression course, and the absence of lymph node metastasis; the average incidence is around age 60. In this case, a young patient presented an ACC tumor with lung and bone metastasis. Due to the rarity, there is no definitive consensus regarding the ideal treatment, with the literature referring to the choice of mastectomy. Conclusions: Although malignant breast neoplasms and nonspecial subtypes, such as ductal and triple negative, have a poor prognosis, breast carcinoma of this aforementioned type has a favorable prognosis. The search for driver mutations in cancers of special types, together with the advances in genetic medicine, allows satisfactory results with target-specific treatments.

Introduction: Secretory carcinoma of the breast is a rare disease, accounting for approximately 0.15% of breast cancer cases. This entity was first described in a child in 1966. However, one of the largest case series with SEER data, encompassing a total of 190 patients, showed that the median age at diagnosis was 56 years, and it can affect both sexes, being much more common in women. In this same series, 58% and 40% of patients were positive for estrogen and progesterone hormone receptors, respectively. Most cases (86.86%) were well to moderately differentiated tumors without lymph node involvement. Older patients had a worse prognosis. In general, the secretory breast carcinoma has a more indolent course with excellent prognosis. The treatment is based mainly on surgery, followed by radiation therapy. The role of chemotherapy and hormone therapy in these cases is not yet well established in the literature. Parallel to basal-like breast cancer, the indolent clinical course as well as prolonged survival seems opposite to that of common triple-negative breast cancer. In most cases of secretory breast carcinoma, reaching a 92% positivity rate, there is a fusion of the ETV6-NTRK3 genes, activating aberrant cell proliferation pathways. Studies with NTRK inhibitors are being developed and will bring this therapeutic possibility soon. Due to the rarity of secretory carcinoma of the breast, notably in men, we report the case of a young man with this neoplasm. A.S.R., 20 years old, male, from Guaraciaba do Norte (CE), white, single, telecommunications technician, reported that he noticed a painless, small, stable nodule in his left breast in 2012. He did not seek medical help at the time. In June 2021, the patient suffered trauma to her left breast during a soccer match. After this event, she noticed a considerable growth of a nodule in the left breast, which became painful to palpation. He then sought medical attention in a health center and underwent an ultrasonography of the left breast in August 2021, which detected a hypoechoic nodular image, oval, with well-defined limits, and regular contours, measuring 16.1×9.6×13.7 cm, 1.8 mm away from the skin, without vascularization inside the nodule (CATEGORY: BIRADS 3); little amount of stromal and glandular tissue were observed. A core biopsy of the nodule, performed in August 26, 2022, showed atypical epithelial proliferation. Immunohistochemistry was compatible with hypersecretory atypical epithelial proliferation. After the diagnosis of secretory carcinoma of the breast, the patient was referred to the Haroldo Juaçaba Hospital, a reference hospital in oncologic treatment in the North/Northeast of Brazil, where he underwent a slide review and immunohistochemistry, which confirmed invasive carcinoma of the secretory type of breast. Staging CT scans and bone scintigraphy were performed in September 2021. Chest CT showed a nodule with irregular contours and contrast medium concentration in the left breast, in close contact with the retropectoral musculature, measuring 19×10×12 mm, in addition to adenomegaly in the right axillary region (levels I and II), measuring up to 38×27 mm. There were no other relevant findings, with no evidence of secondary disease in the abdomen and bones. Investigation of right axillary adenomegaly with core biopsy continued and was negative for neoplasia. Histological picture and immunohistochemical profile were compatible with mixed lymphoid hyperplasia, follicular, and interfollicular. In December 28, 2022, the patient underwent a left mastectomy with sentinel lymph node biopsy, which revealed a secretory invasive carcinoma, measuring 1.9×1.5 cm, grade I, lymphovascular invasion, and negative margins, no lymph nodes were involved, nipple with compromised dermis and intraductal extension. Pathological staging: pT1c pN0 (sn-). The patient is currently on adjuvant systemic treatment (chemotherapy) with good tolerance.