Bi-annual newsletter

November 2015

EMTICS cohort studies: enrolment complete!

We have now completed the 4^th year of EMTICS. The enrolment time for the two main cohort studies has now been completed. We have recruited above target for the COURSE study, with 719 children who have entered the study, a fantastic achievement that results from the general dedication and commendable hard work performed at all clinical sites! The first part of 2016 will be dedicated to complete the follow up of these patients: during a period of more than one and a half year, these children fill out weekly diaries in which they document any changes in tic severity alongside with the possible occurrence of infections and social stress. There are also scheduled hospital visits during which throat swabs, hair samples and blood samples are taken. The ultimate goal is to link environmental factors and changes in biomarkers to tic exacerbations. So far, we have recorded already 162 expedited visits, which indicate the possible presence of an exacerbation event in this cohort, and more are expected in the remaining follow-up period. Things continue to look very promising for this cohort study!

Enrolment has been completed also for the ONSET study, which deals with the monitoring of children at risk of developing tics. Overall, 263 children were recruited into this study, and at least 37 new onset events have been captured, and, like for the COURSE study, more are expected in the remaining follow-up period. Although below target, the cohort recruited so far represents, to the best of our knowledge, the only prospective series of children with genetic predisposition to tics who have been followed up prior to tic onset.

Additional efforts are being made to face the difficulties in recruitment for the TREATMENT study, our clinical antibiotic trial in which children with an already established chronic tic disorder and non-symptomatic throat carriage of group A Streptococcus are being enrolled. In order to increase the recruitment, University of Roma submitted an amendment to the European Commission, planning the extension of the population considered for inclusion by admitting to the enrolment also children who do not participate to the COURSE study, and shortening the trial duration. According to the changes introduced by this amendment, University of Roma centre postponed the date of possible enrolment of the last patient to 20^th September 2016. A recalculation of the target number that should be achieved in order to obtain valuable outcome data has been completed, with the current target set up at 46 patients. University of Roma has also arranged the third provision of active and placebo drugs to participating centres.

The analyses on the ‘bad bug’ continue

The Microbiology team of the Istituto Superiore di Sanità (ISS) in Rome continues its work in order to address one of the core hypotheses of the study: are the onset and/or exacerbation of tic and comorbid obsessive-compulsive disorders in children associated with increased preceding occurrence of Streptococcus pyogenes (group A streptococcus, GAS) exposure or with an infection caused by specific molecular subtypes of this germ? This team is working on the microbiological characterization of GAS strains isolated from the throat swabs of patients during the ONSET, COURSE and TREATMENT studies. The aim is to assess if specific clones and/or their repertoire of virulence factors can be associated to the insurgence or recrudescence of tic symptoms. GAS strains are isolated from the throat of children, swabbed both at the time of recruitment and according to a planned visit schedule by clinical partners. Throat swabs are processed by the microbiology laboratories following a common validated microbiological protocol.

As the number of isolated GAS strains increases (at present, the Istituto Superiore di Sanità has collected 64 strains from ONSET and 156 strains from COURSE), some interesting aspects are appearing, although data require final confirmation, and should therefore be treated with caution: 1) exposure to GAS is more common in both ONSET and COURSE children in comparison to the exposure reported in the general paediatric population, particularly among subjects with tics in the lower age range (3-6 years); 2) specific serotypes of GAS were over-represented in the overall group of GAS strains isolated from children presenting the most severe tic symptoms (according to the Clinical Global Impression Scale); likewise, specific variants of the gene coding for a particular ‘toxic’ protein of GAS, the superantigen speC, were also over-represented in strains isolated from more severe patients.

Basic science work on mouse continues with success

One of the aims of EMTICS is to elucidate the complex aetiology of chronic tic disorders. One of the key hypotheses of EMTICS is that repeated encounters with GAS may facilitate the manifestation of tics. Within the array of disciplines that contribute to the project built around this hypothesis, EMTICS is also leveraging experimental animal models. To complement human studies with laboratory animals, the ISS team has selected a group of infection-prone mice (the rodent analogue of a kid with frequent sore throat episodes) and repeatedly administered them with a homogenate preparation from GAS. Afterwards, our colleagues studied whether the behaviour of these mice resembled human tics.

The first animal model study has been completed by the ISS team and published (M. Proietti Onori et al. 2014; doi: 10.1016/j.bbr.2014.03.023). In a subsequent study, our colleagues exposed the infection-prone mouse strain (SJL/J) to repeated immunizations with GAS. Behavioural and brain immunohistochemical data suggested that GAS immunizations may result in a Tourette’s Syndrome-like phenotype. These data have been recently published in an open access journal, accessible from any internet-connected device around the globe (Macrì et al., 2015; doi:10.1038/srep13257). Leveraging this finding, the ISS team is now testing whether various forms of life stress (physiological and psychosocial) modulate these phenomena. An ongoing study demonstrated that precocious environmental stressors are capable of modulating the course of tic-like symptoms in rodents repeatedly exposed to GAS. Furthermore, this study suggests that the regulatory effects of precocious stressors are likely mediated by the immune system. This provides further evidence that the immune system may play a pivotal role in TS. An additional article has been published widening the debate on possible therapeutic strategies in tic disorders. This study investigated whether an indirect cannabinoid agonist (URB597) may reduce the exhibition of the selective serotonin 5-HT2 receptor agonist (DOI)-induced head twitch responses in mice (C. Ceci , M. Proietti Onori, S. Macrì, G. Laviola;  doi: 10.1007/s12640-014-9510-z. 2014.12.17).

Stay tuned…

Genetic studies in EMTICS

The aim of the genetic workpackage of EMTICS is to unravel genes and gene pathways that may be implicated in the pathogenesis of tics and related comorbidities in GTS patients, as well as to unveil gene-environment interactions that may influence disease onset or its clinical course. For this purpose, our partners at the Democritus University of Thrace, at Alexandropoulis, Greece, are creating a biobank consisting of DNA and RNA samples from a large number of subjects from the COURSE and ONSET cohorts. In cooperation with 16 clinical sites, they have completed the collection and process of 613 COURSE and 20 ONSET DNA samples. These samples will be used for a genome-wide association study (GWAS), targeting more than 710,000 polymorphic markers (i.e. SNPs), and will be also useful to explore potential gene-gene and gene-environment interactions that may influence the pathogenesis of tics and obsessive-compulsive symptoms. A pipeline to analyze GWAS data and methods for population stratification correction, typically introduced in such type of analysis, has been established. As a next step, our colleagues are planning to utilize data from the large-scale TSAICG GWAS to perform meta-analyses combined with the EMTICS data, in order to increase the power of such analyses in an effort to dissect the genetic basis of the complex GTS phenotype. In parallel, RNA samples are being collected to perform genome-wide gene expression analyses, aiming to reveal the genetic pathways that influence the clinical course of tic disorders (i.e. disease onset) and are activated upon symptom exacerbations (i.e. exacerbation versus remission). To this end, samples from the majority of the target ONSET cohort at baseline and nearly half of the samples for the COURSE study upon their first tic exacerbation have been collected. While collecting RNA samples, our colleagues are developing computational tools and test statistical methods to analyze whole-transcriptome data, using analogous datasets via external collaborations.

Exploring psychosocial stress

Workpackage 8 of EMTICS investigates the influence of stress on the tic fluctuations. Our colleagues at the University of Dresden are coordinating this work and completed their first experimental study, which explored the effects of the Trier Social Stress Test for children (TSST-C) on tic frequency in 31 children and adolescents with tic disorders. A relaxation and a concentration situation served as control conditions. Considering the suggestion that stress has an influence on the ability to suppress tics, patients received two different instructions for each situation: 1) to suppress their tics and 2) to “tic freely”. Physiological measures of stress were measured throughout the whole experiment. The TSST-C elicited a clear stress response with elevated levels of saliva cortisol, increased heart rate and higher number of skin conductance responses. With regard to tic frequency, our colleagues observed an interaction effect between situation and instruction: during relaxation and concentration the instruction to suppress tics reduced the number of tics, while during stress the number of tics was low regardless of the instruction. Our study suggests that stress might result in a situational decrease of tic frequency. Different from previous findings, stress did not reduce the ability to suppress tics. Discrepancies to previous self-report studies might be explained by the irritability of subjective tic ratings. Furthermore the analysis of the hair strains is in process. With these additional stress markers we expect to obtain information about the association between stress and tics in a long-term perspective.

Data management

Data management of the study continues to be successful. Data input in the web database has been successful, and information collected from almost all enrolled patients has now been inserted into the database. Storage of biological specimens for immunological measures as well as for DNA and gene expression analyses is moving along, and important decisions have been taken in respect to the choice of gene expression and genotyping analyses platforms.

Dissemination initiatives in 2015 so far

In the second half of 2015 two important dissemination events were held: the 1st World Congress on Tourette Syndrome and Tic Disorders in London and the EMTICS General Assembly meeting in Bari. The first was a successfully event and during the second one important decisions were taken about biological samples and scientific results’ management.

• The conference “Therapy in Child and Adolescent Neuropsychiatry” next April 2016 in Catania, Italy, organized in collaboration with the Italian Society of Child and Adolescent Neuropsychiatry (SINPIA);

• The meeting “Therapies in Childhood Tic Disorders” in May 2016 in Rome, organized by Professor Francesco Cardona, University of Rome La Sapienza;

• The 2016 Annual Meeting of the European Society for the Study of Tourette Syndrome for the Study of Tourette Syndrome and 9^th European Conference on Tourette Syndrome and Tic Disorders will take place on June 8-11, 2016 in Warsaw, Poland. Advances in Neurobiology, Neurophysiology and Imaging, Genetic, Therapy and Clinical Management of Tic Disorders will be discussed;

• The EMTICS General Assembly will be in Lisboa (portugal) in November 2016.

The EMTICS Steering Committee meeting will meet in Leiden (The Netherlands) in May 2016. The next bi-annual letter will be published shortly thereafter in that month.