The alliance between genetic biobanks and patient organisations: the experience of the telethon network of genetic biobanks

Background: Rare diseases (RDs) are often neglected because they affect a small percentage of the population (6-8 %), which makes research and development of new therapies challenging processes. Easy access to high-quality samples and associated clinical data is therefore a key prerequisite for biomedical research. In this context, Genetic Biobanks are critical to developing basic, translational and clinical research on RDs. The Telethon Network of Genetic Biobanks (TNGB) is aware of the importance of biobanking as a service for patients and has started a dialogue with RD-Patient Organisations via promotion of dedicated meetings and round-tables, as well as by including their representatives on the TNGB Advisory Board. This has enabled the active involvement of POs in drafting biobank policies and procedures, including those concerning ethical issues. Here, we report on our experience with RD-Patient Organisations who have requested the services of existing biobanks belonging to TNGB and describe how these relationships were established, formalised and maintained. Results: The process of patient engagement has proven to be successful both for lay members, who increased their understanding of the complex processes of biobanking, and for professionals, who gained awareness of the needs and expectations of the people involved. This collaboration has resulted in a real interest on the part of Patient Organisations in the biobanking service, which has led to 13 written agreements designed to formalise this process. These agreements enabled the centralisation of rare genetic disease biospecimens and their related data, thus making them available to the scientific community. Conclusions: The TNGB experience has proven to be an example of good practice with regard to patient engagement in biobanking and may serve as a model of collaboration between disease-oriented Biobanks and Patient Organisations. Such collaboration serves to enhance awareness and trust and to encourage the scientific community to address research on RDs

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders.Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes.Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs. The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology.Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% (n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage.Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene

Telethon Network of Genetic Biobanks: a key service for diagnosis and research on rare diseases

Several examples have always illustrated how access to large numbers of biospecimens and associated data plays a pivotal role in the identification of disease genes and the development of pharmaceuticals. Hence, allowing researchers to access to significant numbers of quality samples and data, genetic biobanks are a powerful tool in basic, translational and clinical research into rare diseases. Recently demand for well-annotated and properly-preserved specimens is growing at a high rate, and is expected to grow for years to come. The best effective solution to this issue is to enhance the potentialities of well-managed biobanks by building a network.Here we report a 5-year experience of the Telethon Network of Genetic Biobanks (TNGB), a non-profit association of Italian repositories created in 2008 to form a virtually unique catalogue of biospecimens and associated data, which presently lists more than 750 rare genetic defects. The process of TNGB harmonisation has been mainly achieved through the adoption of a unique, centrally coordinated, IT infrastructure, which has enabled (i) standardisation of all the TNGB procedures and activities; (ii) creation of an updated TNGB online catalogue, based on minimal data set and controlled terminologies; (iii) sample access policy managed via a shared request control panel at web portal. TNGB has been engaged in disseminating information on its services into both scientific/biomedical - national and international - contexts, as well as associations of patients and families. Indeed, during the last 5-years national and international scientists extensively used the TNGB with different purposes resulting in more than 250 scientific publications. In addition, since its inception the TNGB is an associated member of the Biobanking and Biomolecular Resources Research Infrastructure and recently joined the EuroBioBank network. Moreover, the involvement of patients and families, leading to the formalization of various agreements between TNGB and Patients' Associations, has demonstrated how promoting Biobank services can be instrumental in gaining a critical mass of samples essential for research, as well as, raising awareness, trust and interest of the general public in Biobanks. This article focuses on some fundamental aspects of networking and demonstrates how the translational research benefits from a sustained infrastructure

Clostridium difficile: 18 months surveillance at the Niguarda Hospital - Milano

Clostridium difficile is currently one of the most common cause of diarrhea in hospitalized or residents in long term care institutions patients, symptoms of infection ranging from diarrhea to pseudo-membranous colitis and toxic megacolon.The disease is due to the production of enterotoxin A, cytotoxin B or binary toxin. The emergence of hypervirulent new strains showing a tcdC regulatory gene deletion (ribotype 027) has been observed in recent years. Stool or rectal swab specimens were screened using automated real-time multiplex PCR (GeneXpert, Cellbio) to detect the presence of toxins producing (B toxin, binary toxin and tcdC gene deletion) C. difficile. All positive samples were cultured in order to isolate the causing infection strains.The binary toxin and/or tcdC deletion producing strains were genotyped using Multilocus Sequence Typing (MLST). MLST compares the intragenic sequences of seven housekeeping genes and provides a unique combination of alleles; to each combination is then assigned a Sequence Typing (ST). In the period January 2010 – June 2011, 3681 samples from 2234 patients were analyzed. Four hundred seventy-three patients (21.2%) were positive for the presence of C. difficile, 34 (7.2%) of there were also positive for binary toxin and 3 were positive for the tcdC gene deletion (suspected C. difficile NAP027).All the strains having tcdC gene deletion, showes ST3 typing by MLST method.These strains were isolated during the same period. The three patiens were epidemiologically linked: the patient A was hospitalized in the same room of patient B, while patient C was managed by the same team care of patient A.The spread ofsuch C. difficile strains is at the present very low in Italy. Our data confirm the poor prevalence of ribotype NAP027 and the usefulness of a presumptive diagnosis to implement immediately appropriate control measures

Assessing Population Trends of Species with Imperfect Detection: Double Count Analyses and Simulations Confirm Reliable Estimates in Brown Frogs

Most animal species are detected imperfectly and overlooking individuals can result in a biased inference of the abundance patterns and underlying processes. Several techniques can incorporate the imperfect detection process for a more accurate estimation of abundance, but most of them require repeated surveys, i.e., more sampling effort compared to single counts. In this study, we used the dependent double-observer approach to estimate the detection probability of the egg clutches of two brown frog species, Rana dalmatina and R. latastei. We then simulated the data of a declining population at different levels of detection probability in order to assess under which conditions the double counts provided better estimates of population trends compared to naïve egg counts, given the detectability of frog clutches. Both species showed a very high detection probability, with average values of 93% for Rana dalmatina and 97% for R. latastei. Simulations showed that not considering imperfect detection reduces the power of detecting population trends if detection probability is low. However, at high detection probability (>80%), ignoring the imperfect detection does not bias the estimates of population trends. This suggests that, for species laying large and easily identifiable egg clutches, a single count can provide useful estimates if surveys are correctly timed

Engineering of the spin on dopant process on silicon on insulator substrate

We report on a systematic analysis of phosphorus diffusion in silicon on insulator thin film via spin-on-dopant process (SOD). This method is used to provide an impurity source for semiconductor junction fabrication. The dopant is first spread into the substrate via SOD and then diffused by a rapid thermal annealing process. The dopant concentration and electron mobility were characterized at room and low temperature by four-probe and Hall bar electrical measurements. Time-of-flight-secondary ion mass spectroscopy was performed to estimate the diffusion profile of phosphorus for different annealing treatments. We find that a high phosphorous concentration (greater than 1020 atoms cm-3) with a limited diffusion of other chemical species and allowing to tune the electrical properties via annealing at high temperature for short time. The ease of implementation of the process, the low cost of the technique, the possibility to dope selectively and the uniform doping manufactured with statistical process control show that the methodology applied is very promising as an alternative to the conventional doping methods for the implementation of optoelectronic devices. </p

Isolated limb dystonia as presenting feature of Parkin disease

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A family with paroxysmal nonkinesigenic dyskinesias (PNKD): Evidence of mitochondrial dysfunction

Introduction: Paroxysmal nonkinesigenic dyskinesia (PNKD) is a rare movement disorder characterized by sudden attacks of involuntary movements. Familial PNKD is an auto- somal dominant trait, caused by mutations in the myofibrillogenesis regulator 1 ( MR-1 ) gene on chromosome 2q35. Three different mutations have been described; all of them reside in the N-terminal region common to isoforms L and S, that has been suggested to code for a mitochondrial targeting sequence, necessary for the correct sub-cellular locali- zation of the protein into mitochondria. Methods: We report on four patients of the same family, affected by PNKD. Skin fibroblasts were used to analysed oxygen consumption and to measure mitochondrial matrix calcium response after agonist stimulation. Mitotracker-based visualization was also used to assess fragmentation of the mitochondrial network. Results: the paroxysmal movements were dystonic in two patients and dystonic/choreiform in the other ones; in three cases the symptoms started in one limb and then generalized, while in one case remained focal. Three had a very early onset, within the first two years of life. The frequency of episodes showed a great variability, ranging from 2 times a day to 3 times a year, while the duration of the attacks ranged from 2 min to 1,5 h, always with sudden onset and end and complete recover in between. All affected subjects harbored a heterozygous C to T substitution in MR-1 , causing an Ala9Val amino acid change in the N- terminal region