Manuel Posada

RD-Connect: An Integrated Platform Connecting Databases, Registries, Biobanks and Clinical Bioinformatics for Rare Disease Research

ABSTRACTResearch into rare diseases is typically fragmented by data type and disease. Individual efforts often have poor interoperability and do not systematically connect data across clinical phenotype, genomic data, biomaterial availability, and research/trial data sets. Such data must be linked at both an individual-patient and whole-cohort level to enable researchers to gain a complete view of their disease and patient population of interest. Data access and authorization procedures are required to allow researchers in multiple institutions to securely compare results and gain new insights. Funded by the European Union’s Seventh Framework Programme under the International Rare Diseases Research Consortium (IRDiRC), RD-Connect is a global infrastructure project initiated in November 2012 that links genomic data with registries, biobanks, and clinical bioinformatics tools to produce a central research resource for rare diseases.

The TREAT‐NMD Duchenne Muscular Dystrophy Registries: Conception, Design, and Utilization by Industry and Academia

Duchenne muscular dystrophy (DMD) is an X‐linked genetic disease, caused by the absence of the dystrophin protein. Although many novel therapies are under development for DMD, there is currently no cure and affected individuals are often confined to a wheelchair by their teens and die in their twenties/thirties. DMD is a rare disease (prevalence <5/10,000). Even the largest countries do not have enough affected patients to rigorously assess novel therapies, unravel genetic complexities, and determine patient outcomes. TREAT‐NMD is a worldwide network for neuromuscular diseases that provides an infrastructure to support the delivery of promising new therapies for patients. The harmonized implementation of national and ultimately global patient registries has been central to the success of TREAT‐NMD. For the DMD registries within TREAT‐NMD, individual countries have chosen to collect patient information in the form of standardized patient registries to increase the overall patient population on which clinical outcomes and new technologies can be assessed. The registries comprise more than 13,500 patients from 31 different countries. Here, we describe how the TREAT‐NMD national patient registries for DMD were established. We look at their continued growth and assess how successful they have been at fostering collaboration between academia, patient organizations, and industry.

Fish consumption patterns and hair mercury levels in children and their mothers in 17 EU countries

The toxicity of methylmercury (MeHg) in humans is well established and the main source of exposure is via the consumption of large marine fish and mammals. Of particular concern are the potential neurodevelopmental effects of early life exposure to low-levels of MeHg. Therefore, it is important that pregnant women, children and women of childbearing age are, as far as possible, protected from MeHg exposure. Within the European project DEMOCOPHES, we have analyzed mercury (Hg) in hair in 1799 mother-child pairs from 17 European countries using a strictly harmonized protocol for mercury analysis. Parallel, harmonized questionnaires on dietary habits provided information on consumption patterns of fish and marine products. After hierarchical cluster analysis of consumption habits of the mother-child pairs, the DEMOCOPHES cohort can be classified into two branches of approximately similar size: one with high fish consumption (H) and another with low consumption (L). All countries have representatives in both branches, but Belgium, Denmark, Spain, Portugal and Sweden have twice as many or more mother-child pairs in H than in L. For Switzerland, Czech Republic, Hungary, Poland, Romania, Slovenia and Slovakia the situation is the opposite, with more representatives in L than H. There is a strong correlation (r=0.72) in hair mercury concentration between the mother and child in the same family, which indicates that they have a similar exposure situation. The clustering of mother-child pairs on basis of their fish consumption revealed some interesting patterns. One is that for the same sea fish consumption, other food items of marine origin, like seafood products or shellfish, contribute significantly to the mercury levels in hair. We conclude that additional studies are needed to assess and quantify exposure to mercury from seafood products, in particular. The cluster analysis also showed that 95% of mothers who consume once per week fish only, and no other marine products, have mercury levels 0.55 μg/g. Thus, the 95th percentile of the distribution in this group is only around half the US-EPA recommended threshold of 1 μg/g mercury in hair. Consumption of freshwater fish played a minor role in contributing to mercury exposure in the studied cohort. The DEMOCOPHES data shows that there are significant differences in MeHg exposure across the EU and that exposure is highly correlated with consumption of fish and marine products. Fish and marine products are key components of a healthy human diet and are important both traditionally and culturally in many parts of Europe. Therefore, the communication of the potential risks of mercury exposure needs to be carefully balanced to take into account traditional and cultural values as well as the potential health benefits from fish consumption. European harmonized human biomonitoring programs provide an additional dimension to national HMB programs and can assist national authorities to tailor mitigation and adaptation strategies (dietary advice, risk communication, etc.) to their countrys specific requirements.

The need for worldwide policy and action plans for rare diseases

There are more than 6000 rare diseases (defined as affecting <5/10 000 individuals in Europe, <200 000 people in the United States). The rarity can create problems including: difficulties in obtaining timely, accurate diagnoses; lack of experienced healthcare providers; useful, reliable and timely information may be hard to find; research activities are less common; developing new medicines may not be economically feasible; treatments are sometimes very expensive; and in developing countries, the problems are compounded by other resource limitations. Emphasis is required to support appropriate research and development leading to better prevention, diagnosis and treatments of rare diseases. Notably, clinical trials using already existing drugs may result in new, affordable, treatment strategies. Moreover, rare diseases may teach us about common disorders.

La red de investigación "infancia y medio ambiente"(Red INMA): Protocolo de estudio

Cada vez existe mayor evidencia de la influencia de la dieta y de la exposicion a dosis bajas de toxicos durante la etapa prenatal y primera infancia sobre la salud y el bienestar en etapas posteriores de la vida. Siguiendo las recomendaciones de la OMS y de la Union Europea en el ano 2003 se constituyo la Red de Investigacion Cooperativa Infancia y Medio Ambiente para estudiar los efectos del medio ambiente y la dieta en el desarrollo fetal e infantil en diversas zonas geograficas en Espana. La Red integra diversos grupos multidisciplinares de investigacion y esta constituida por seis cohortes, tres preexistentes y tres de novo, que seguiran de forma prospectiva a 3.600 mujeres embarazadas, desde el inicio del embarazo hasta los 4-6 anos del nino. Los objetivos generales de la red son: (1) Describir la exposicion individual a toxicos ambientales durante la gestacion y la primera infancia. (2) Evaluar los efectos de la exposicion a toxicos y de la dieta en el desarrollo fetal e infantil. (3) Evaluar la interaccion entre factores toxicos, nutricionales y geneticos en el desarrollo fetal e infantil. El seguimiento se realiza en cada trimestre de la gestacion, al nacimiento, al ano y hasta los cuatro o seis anos del nino. La informacion se recoge mediante cuestionarios, datos clinicos, exploracion fisica, ecografias, biomarcadores y mediciones ambientales. En este trabajo se presentan las caracteristicas generales de la red y se describe la situacion actual de cada una de las cohortes.

Cytokine mRNA expression in lung tissue from toxic oil syndrome patients: a TH2 immunological mechanism

In 1981, an epidemic occurred in Spain, toxic oil syndrome (TOS), in people who consumed rapeseed oil denatured with 2% aniline, and it was one of the largest intoxication epidemics ever recorded. In 1989, a similar disease, eosinophilia-myalgia syndrome (EMS) was reported in the USA and was associated with the ingestion of L-tryptophan. The pathologic findings in TOS showed primary endothelial injury, with cell proliferation and perivascular inflammatory infiltrates. Immunologic mechanisms have presumably been operative in the pathogenesis and perpetuation of TOS. Our previous findings pointed to a T-cell activation during acute phase of the disease. In order to analyze which T-cell subset is involved on TOS, we have developed an mRNA extraction procedure from paraffin-embedded lung tissues in patients with pulmonary involvement. We analyzed mRNA expression from different cytokines (IL-1, IL-2, IL-4, IL-5, IFN-gamma, GM-CSF) and CD25 (interleukin 2 receptor) and CD23 (low affinity IgE receptor), using RT-PCR technique. In lung tissues from these patients a T-cell activation was observed. We found a significant increase in Th1 (P = 0.006) and Th2 (P = 0.003) cytokine profile in TOS patients with respect to controls. The increment in TH2 response with respect to TH1 is significant (P = 0.03) in TOS lung specimens. Non-significant differences were obtained in other cytokines and receptors studied as IL-1, CD25, CD23 and GM-CSF. Data presented in this paper are the first clear evidence that an immunological mechanism is directly implicated in this illness.

Immunological basis of toxic oil syndrome (TOS)

The toxic oil syndrome (TOS), a multisystemic disease, that occurred in Spain in 1981, was caused by the ingestion of rapeseed oil denatured with 2% aniline. Due to the clinical course of the disease, immunopathological mechanisms have been suspected but a direct connection was never demonstrated. To analyse this possibility, we determined several immunological parameters in the sera of patients with TOS and without the disease, using a case-control design: total immunoglobulins, IgG and IgE antibodies against different toxic agents (oleylanilide, aniline, linoleyl-anilide, and 3-phenylaminopropane-1-2-diol), autoantibodies, cytokines (IL-4, IL-6, TNF, GM-CSF) and soluble receptors (sCD23 and sIL-2R). We detected high levels of sIL-2R in TOS patients compared to controls (P < 0.0001). A higher levels of sCD23 and IgE were also found. In addition, the response to oleyl-anilide of peripheral blood lymphocytes from TOS patients was studied and a significant proliferative response in 30% of TOS patients versus 5% controls was observed. Our data support the implication of the immune system in the acute phase of TOS, with a possible activation of T-cells and release of cytokines, that could explain some of the clinical findings in this phase of the disease.

Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid

BackgroundSevere Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. It is usually related to PI*Z alleles, and less frequent to rare and null (QO) alleles. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema.MethodsA family with severe AAT deficiency was analyzed to achieve genetic diagnosis. The complete exons and introns of the SERPINA1 gene were sequenced and transcriptional analysis by RT-PCR was performed to characterize the effect of splicing variants found in the patients. In addition, a minigene MGserpa1_ex1b-1c was cloned into the pSAD vector to in vitro investigate the independent impact of variants on splicing process.ResultsWe report a new identified null allele (PI*QOMadrid) in two adult siblings with practically no detectable serum AAT. The PI*QOMadrid allele consist of a duplication of the thymine (T) in position +2 of the donor splice site of exon 1C (+2dupT). In these two subjects, PI*QOMadrid occurred in compound heterozygote combination with the previously described variant PI*QOPorto. Both QOMadrid and QOPorto variants are located very close together in a regulatory region of the SERPINA1 gene. Analysis of transcripts revealed that QOMadrid variant prevented the expression of transcripts from exon 1C, and then normally spliced RNA products are not expected in the liver of these patients. In addition, aberrant splicing patterns of both variants were clearly distinguished and quantified by functional in vitro assays lending further support to their pathogenicity.ConclusionFinding pathogenic mutations in non-coding regions of the SERPINA1 highlight the importance that regulatory regions might have in the disease. Regulatory regions should be seriously considered in discordant cases with severe AAT deficiency where no coding mutations were found.

RARE-Bestpractices: a platform for sharing best practices for the management of rare diseases

Over the last decade the European Union has been coordinating actions addressing various aspects of rare diseases and has funded several cross-border research projects. Recently has initiated the biggest rare disease international collaborative effort by launching the International Rare Diseases Research Consortium (IRDiRC). RARE-Bestpractices is one of the more than 100 collaborative research projects on rare diseases funded under the Seventh Framework Programme for Research and Technological Development (FP7; 2007-2013) (1). As a wide, open and inclusive network, RARE-Bestpractices will build on the knowledge of the experts in rare disease research area and experts in guideline development and health technology assessment area, brought together, for the first time, from academic institutions, agencies, organizations, patient advocacy groups, governmental bodies. The project aims at building a platform to collect and exchange information on best practices for the management of rare diseases; to identify relevant research needs; to promote the development of high quality guidelines; and to contribute in making patients, health professionals and policy makers “informed guideline users”. Besides, RARE-Bestpractices will intend to define the extent to which conclusions from cost-effectiveness analyses for pharmaceuticals are accounted for and implemented in guidelines across a range of countries.

A pilot study on the feasibility of European harmonized Human Biomonitoring: Strategies towards a common approach, challenges and opportunities

In 2004 the European Commission and Member States initiated activities towards a harmonized approach for Human Biomonitoring surveys throughout Europe. The main objective was to sustain environmental health policy by building a coherent and sustainable framework and by increasing the comparability of data across countries. A pilot study to test common guidelines for setting up surveys was considered a key step in this process. Through a bottom-up approach that included all stakeholders, a joint study protocol was elaborated. From September 2011 till February 2012, 17 European countries collected data from 1844 mother-child pairs in the frame of DEMOnstration of a study to COordinate and Perform Human Biomonitoring on a European Scale (DEMOCOPHES).(1) Mercury in hair and urinary cadmium and cotinine were selected as biomarkers of exposure covered by sufficient analytical experience. Phthalate metabolites and Bisphenol A in urine were added to take into account increasing public and political awareness for emerging types of contaminants and to test less advanced markers/markers covered by less analytical experience. Extensive efforts towards chemo-analytical comparability were included. The pilot study showed that common approaches can be found in a context of considerable differences with respect to experience and expertize, socio-cultural background, economic situation and national priorities. It also evidenced that comparable Human Biomonitoring results can be obtained in such context. A European network was built, exchanging information, expertize and experiences, and providing training on all aspects of a survey. A key challenge was finding the right balance between a rigid structure allowing maximal comparability and a flexible approach increasing feasibility and capacity building. Next steps in European harmonization in Human Biomonitoring surveys include the establishment of a joint process for prioritization of substances to cover and biomarkers to develop, linking biomonitoring surveys with health examination surveys and with research, and coping with the diverse implementations of EU regulations and international guidelines with respect to ethics and privacy.