Ricerca di nuove varianti geniche associate alle malattie infiammatorie croniche intestinali

2013/20142013/2014Le malattie infiammatorie croniche intestinali (MICI), sono un gruppo di malattie eterogenee ad eziologia multifattoriale. Sono caratterizzate da uno stato infiammatorio a carico della mucosa del tratto gastrointestinale e comprendono il Morbo di Crohn (MC), la Rettocolite ulcerosa (RCU) e la Colite indeterminata (CI) i cui quadri istopatologici differiscono tra loro per tipo di lesione, localizzazione della malattia e complicanze associate. Le MICI insorgono tipicamente durante l’adolescenza o in età adulta come il risultato della combinazione di tutti i fattori predisponenti che concorrono in egual misura nella determinazione della malattia. L’insorgenza delle MICI può avvenire anche in età molto precoce, entro i 10 anni ma anche entro i 2 anni e in maniera ancora più grave. In questi casi di esordio precoce si ipotizza che il peso maggiore sia da attribuire alla componente genetica piuttosto che a fattori ambientali e microbici. Solitamente i pazienti con esordio precoce sono caratterizzati da un fenotipo malattia più severo e difficilmente controllabile con le terapie convenzionali. Per gli aspetti differenti che si osservano in termini di predisposizione, caratteristiche fenotipiche, fattori coinvolgenti e geni interessati, le MICI possono essere contestualizzate da una parte come malattie multifattoriali e dall’altra come patologie “monogeniche". Nel contesto della multifattorialità, i numerosi studi di associazione son stati importantissimi in quanto hanno individuato numerosi geni relativi a distinte pathway (barriera intestinale, regolazione dell’immunità innata dell’epitelio, autofagia, sistema fagocitario e stress) coinvolte nella patogenesi delle MICI (ad oggi 163 loci). Nel lavoro di dottorato l’attenzione e l’interesse si è focalizzato sullo studio delle MICI ad insorgenza precoce e uno dei primi obiettivi della tesi è stato quello di indagare in 36 pazienti pediatrici, geni noti dalla letteratura per essere associati alla malattia (NOD2, ATG16L1, IL23R, IL10, IL10RA, IL10RB e XIAP), con il fine di identificare una possibile correlazione genotipo-fenotipo. Anche se non è stato possibile identificare un unico filo conduttore che ci ha permesso di correlare il fenotipo dei pazienti ai genotipi individuati, sono state identificate nuove varianti missenso e introniche. Tutte le varianti individuate sono state analizzate da un punto di vista bioinformatico per valutare la predizione di patogenicità: in base alle predizioni ottenute l’attenzione si è focalizzata su due varianti nel gene NOD2 sulle quali sono stati allestiti saggi funzionali per valutare il loro impatto sulla corretta sintesi e funzionamento della proteina. Un importante dato che emerge sempre più spesso dalla letteratura è l’evidenza che lesioni infiammatorie a carico del tratto gastrointestinale e il fenotipo tipico delle MICI, possono presentarsi molto precocemente (entro i 2 anni di vita) come prime o a volte anche come uniche manifestazioni cliniche in un contesto patologico più ampio che sottende allo sviluppo di gravi immunodeficienze (MICI-like). In questi casi le mutazioni a carico del gene malattia sono molto rare e generalmente considerate come mutazioni “private” e causative del fenotipo malattia che si osserva. Nell’ambito delle MICI in un contesto che possiamo definire monogenico, sono stati analizzati pazienti pediatrici con una sintomatologia MICI-like mediante analisi di sequenza di nuova generazione “Whole Exome Sequencing (WES)”. Sono state ricercate specificamente mutazioni in un determinato set di geni accuratamente selezionati (60 geni) in quanto responsabili di patologie monogeniche che presentano, all’esordio della malattia, una sintomatologia MICI-like. L’obiettivo è quello di riuscire ad effettuare in tempi rapidi l’identificazione di mutazioni in specifici geni malattia, per permettere al clinico di diagnosticare altrettanto rapidamente la malattia e poter intraprendere la terapia più adeguata e specifica per ciascun paziente. Così come sono state individuate numerose varianti presenti nei database e note per la loro associazione alle MICI, sono state identificate anche nuove varianti, mai descritte prima in letteratura. Alcune varianti sono state analizzate con saggi funzionali in vitro in modo da poter comprendere il rispettivo effetto sulla proteina. Per testare l’effetto della variante intronica rs104895421 (c.74-7T>A), situata a monte dell’esone 2 del gene NOD2, è stato allestito il saggio del minigene ibrido. L’esperimento ha messo in evidenza che tale sostituzione nucleotidica altera il corretto funzionamento del meccanismo di splicing, provocando, anche se non con una efficienza del 100% l’esclusione dell’esone. Nel contesto di MICI come malattie monogeniche, sono state individuate due importanti mutazioni, in due pazienti con sintomatologia MICI-like ad esordio molto precoce. La prima è una mutazione, ovvero una delezione di due nucleotidi, identificata nel gene XIAP (c.1021_1022delAA fs p.N341fsX7). Questa delezione determina la sintesi di una proteina tronca provocando un’alterazione strutturale della proteina che ne la funzionalità. Il risultato di tale lavoro ha permesso al clinico di fare finalmente la corretta diagnosi e il paziente è stato curato grazie ad un trapianto di midollo. La seconda mutazione degna di interesse è una mutazione missenso identificata in omozigozi nel gene NOD2 (c.G1277A p.R426H), in seguito all’analisi dell’esoma. Dalle indagini funzionali si evince che tale mutazione altera il normale funzionamento del recettore intracellulare NOD2, e quindi potrebbe spiegare il fenotipo malattia osservato nel giovane paziente (“gain of function”). In questo caso, il confronto con il clinico, in base alle evidenze ottenute dai test eseguiti, deve ancora avere luogo ma sarà di fondamentale importanza per fare una diagnosi e iniziare la terapia idonea. Questa tesi ha incrementato, seppur con piccoli tasselli, le conoscenze riguardo alcuni varianti in geni conosciuti dalla letteratura per la loro associazione con le MICI. I risultati ottenuti hanno avuto inoltre un impatto traslazionale molto importante permettendo ai clinici di fare la corretta diagnosi e iniziare la terapia idonea per migliorare la qualità e l’aspettativa di vita del paziente.XXVII CicloXXVII Ciclo198

The diagnostic challenge of very early-onset enterocolitis in an infant with XIAP deficiency

Background: Aggressive course and resistance to treatments usually characterize very early onset inflammatory bowel disease (VEO-IBD). Some VEO-IBD cases are due to monogenic immune defects and can benefit from hematopoietic stem cell transplantation (HSCT). Case presentation: We describe a Caucasian male baby who presented in the first months of life macrophage activation syndrome, followed by intractable colitis, recurrent episodes of fever and mild splenomegaly. After several immunological, genetic and clinical investigations, subsequently a therapeutic attempt with colectomy, analysis of VEO-IBD-associated genes, revealed a causative mutation in XIAP. The genetic diagnosis of a primary immune deficiency allowed curing the boy with hematopoietic stem cell transplantation. Conclusion: Our report, together with novel findings from recent literature, should contribute to increase awareness of monogenic immune defects as a cause of VEO-IBD. Comprehensive genetic analysis can allow a prompt diagnosis, resulting in the choice of effective treatments and sparing useless and damaging procedures

Altered pattern of tumor necrosis factor-Alpha production in peripheral blood monocytes from Crohn's disease basic study

AIM: To evaluate the inflammatory state in Crohn's disease (CD) patients and correlate it with genetic background and microbial spreading. METHODS: By means of flow cytometry, production of tumor necrosis factor-alpha (TNF-\u3b1) was measured in peripheral blood monocytes from patients suffering from CD, ulcerative colitis (UC) and in healthy subjects after stimulation of the NOD2 and TLR pathways. CD patients were genotyped for the three most common NOD2 variants (R702W, G908R and L1007Pfs*2) and basal production of TNF-\u3b1 was correlated to NOD2 genotype. Also, production of TNF-\u3b1 was correlated to plasmatic levels of LPS Binding Protein (LBP), soluble (s) CD14 and to the activity state of the disease. RESULTS: The patients with CD were characterized by a significantly higher monocyte basal expression of TNF-\u3b1 compared with healthy subjects and UC patients, and after stimulation with Pam3CSK4 (ligand of TLR2/1) and MDP-L18 (ligand of NOD2) this difference was maintained, while other microbial stimuli (LPS, ligand of TLR4 and PolyI:C, ligand of TLR3) induced massive activation in CD monocytes as well as in UC and in healthy control cells. There was no significant difference in the production of TNF-\u3b1 between patients who carried CD-associated heterozygous or homozygous variants in NOD2 and patients with wild type NOD2 genotype. Although serum LBP levels have been shown to correlate positively with the state of activity of the disease, TNF-\u3b1 production did not show a clear correlation with either LBP or sCD14 levels in plasma. Moreover, no clear correlation was seen between TNF-\u3b1 production and activity indices in either CD or UC. CONCLUSION: Peripheral monocytes from CD express higher basal and stimulated TNF-\u3b1 than controls, regardless of NOD2 genotype and without a clear correlation with disease activity

Parental Stress, Depression, and Participation in Care Before and During the COVID-19 Pandemic: A Prospective Observational Study in an Italian Neonatal Intensive Care Unit.

Background: Recent studies reported, during the COVID-19 pandemic, increased mental distress among the general population and among women around the childbirth period. COVID-19 pandemic may undermine the vulnerable well-being of parents in Neonatal Intensive Care Units (NICUs). Objective: Our study aimed to explore whether parental stress, depression, and participation in care in an Italian NICU changed significantly over three periods: pre-pandemic (T0), low (T1), and high COVID-19 incidence (T2). Methods: Enrolled parents were assessed with the Parental Stressor Scale in the NICU (PSS:NICU), Edinburgh Postnatal Depression Scale (EPDS), and Index of Parental Participation (IPP). Stress was the study primary outcome. A sample of 108 parents, 34 for each time period, was estimated to be adequate to detect a difference in PSS:NICU stress occurrence level score (SOL) of 1.25 points between time periods. To estimate score differences among the three study periods a non-parametric analysis was performed. Correlation among scores was assessed with Spearman rank coefficient. Results: Overall, 152 parents were included in the study (62 in T0, 56 in T1, and 34 in T2). No significant differences in the median PSS:NICU, EPDS, and IPP scores were observed over the three periods, except for a slight increase in the PSS:NICU parental role sub-score in T2 (T0 3.3 [2.3-4.1] vs. T2 3.9 [3.1-4.3]; p = 0.038). In particular, the question regarding the separation from the infant resulted the most stressful aspect during T2 (T0 4.0 [4.0-5.0] vs. T2 5.0 [4.0-5.0], p = 0.008). The correlation between participation and stress scores (r = 0.19-022), and between participation and depression scores (r = 0.27) were weak, while among depression and stress, a moderate positive correlation was found (r = 0.45-0.48). Conclusions: This study suggests that parental stress and depression may be contained during the COVID-19 pandemic, while participation may be ensured

Alendronate, a double-edged sword acting in the mevalonate pathway

Aminobisphosphonate aledronate is a compound commonly used clinically for the treatment of osteoporosis and other bone diseases, as a result of it preventing bone resorption. However, in previous years it has also been used to obtain cellular and animal models of a rare genetic disorder termed Mevalonate Kinase Deficiency (MKD). MKD is caused by mutations affecting the mevalonate kinase enzyme, in the cholesterol pathway and alendronate can be used to biochemically mimic the genetic defect as it inhibits farnesyl pyrophosphate synthase in the same pathway. Despite evidence in favor of the inhibition exerted on the mevalonate pathway, there is at least one clinical case of MKD in which alendronate improved not only skeletal and bone fractures, as expected, but also MKD clinical features. Based on this finding, the present study assessed the anti‑inflammatory properties of this aminobisphosphonate in vitro. No anti‑inflammatory effects of alendronate were observed in the in vitro experiments. Since MKD lacks specific treatments, these results may assist scientists and physicians in making the decision as to the most suitable choice of therapeutic compounds for this neglected disease

Novel Missense Mutation in the NOD2 Gene in a Patient with Early Onset Ulcerative Colitis: Causal or Chance Association?

Deregulated immune response to gut microflora in genetically predisposed individuals is typical for inflammatory bowel diseases. It is reasonable to assume that genetic association with the disease will be more pronounced in subjects with early onset than adult onset. The nucleotide-binding oligomerization domain containing-2 gene, commonly involved in multifactorial risk of Crohn's disease, and interleukin 10 receptor genes, associated with rare forms of early onset inflammatory bowel diseases, were sequenced in an early onset patient. We identified a novel variant in the NOD2 gene (c.2857A > G p.K953E) and two already described missense variants in the IL10RA gene (S159G and G351R). The new NOD2 missense variant was examined in silico with two online bioinformatics tools to predict the potentially deleterious effects of the mutation. Although cumulative effect of these variations in the early onset of the disease can be only hypothesized, we demonstrated that family information and in silico studies can be used to predict association with the disease

Database tools in genetic diseases research

The knowledge of the human genome is in continuous progression: a large number of databases have been developed to make meaningful connections among worldwide scientific discoveries. This paper reviews bioinformatics resources and database tools specialized in disseminating information regarding genetic disorders. The databases described are useful for managing sample sequences, gene expression and post-transcriptional regulation. In relation to data sets available from genome-wide association studies, we describe databases that could be the starting point for developing studies in the field of complex diseases, particularly those in which the causal genes are difficult to identify

Genetic and immunologic findings in children with recurrent aphthous stomatitis with systemic inflammation

Recurrent aphthous stomatitis with systemic signs of inflammation can be encountered in inflammatory bowel disease, Beh\ue7et's disease (BD), Systemic Lupus Erythematosus (SLE). In addition, it has been proposed that cases with very early onset in childhood can be underpinned by rare monogenic defects of immunity, which may require targeted treatments. Thus, subjects with early onset recurrent aphthous stomatitis receiving a clinical diagnosis of BD-like or SLE-like disease may deserve a further diagnostic workout, including immunologic and genetic investigations

Immunity and Genetics at the Revolving Doors of Diagnostics in Primary Immunodeficiencies

Primary immunodeficiencies (PIDs) are a large and growing group of disorders commonly associated with recurrent infections. However, nowadays, we know that PIDs often carry with them consequences related to organ or hematologic autoimmunity, autoinflammation, and lymphoproliferation in addition to simple susceptibility to pathogens. Alongside this conceptual development, there has been technical advancement, given by the new but already established diagnostic possibilities offered by new genetic testing (e.g., next-generation sequencing). Nevertheless, there is also the need to understand the large number of gene variants detected with these powerful methods. That means advancing beyond genetic results and resorting to the clinical phenotype and to immunological or alternative molecular tests that allow us to prove the causative role of a genetic variant of uncertain significance and/or better define the underlying pathophysiological mechanism. Furthermore, because of the rapid availability of results, laboratory immunoassays are still critical to diagnosing many PIDs, even in screening settings. Fundamental is the integration between different specialties and the development of multidisciplinary and flexible diagnostic workflows. This paper aims to tell these evolving aspects of immunodeficiencies, which are summarized in five key messages, through introducing and exemplifying five clinical cases, focusing on diseases that could benefit targeted therapy

Inflammation profile of four early onset Crohn patients

Crohn disease (CD) is a multifactorial disorder affecting mainly young adults. Sometimes, however, it can present in the first year of life (Early onset Crohn disease (EOCD)) showing an unpredictable course and can often be more severe than at older ages. Some cases have been associated to an underlying primary immunodeficiency such as IL10R deficiency. We studied the functional response to IL-10 and the genotype of IL-10 receptor in four patients with early onset crohn-like colitis. We found an IL10R variant, which may be associated with a decreased response to the cytokine in one patient. Further studies to determine its pathogenic effect should be performed. In addition IL-10 mediated inhibition of LPS-induced TNFα expression was measured in patient's monocytes