Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness.

PURPOSE: Several hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology. METHODS: Exome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions. RESULTS: We identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1, were also found. The remaining well-characterized unsolved patients (48%) need further investigation. CONCLUSION: Using our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes.

RD-Connect: an integrated platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research.

Research 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.

Loss of retinal capillary vasoconstrictor response to Endothelin-1 following pressure increments in living isolated rat retinas.

Increased intraocular pressure (IOP) is a major risk factor for glaucoma, and its contribution to neuronal damage appears multi-factorial. An open issue is whether pressure effects on blood vessels contribute to neuronal damage. In particular, little is known about pressure effects on capillaries, which are the site of most metabolic exchange in the retina, but cannot be easily visualized in vivo. To address this issue, here we have imaged retinal capillaries in acutely isolated living rat retinas, and measured alterations in capillary viability, caliber and response to vasoactive stimuli after controlled pressure stimuli. We found that capillary viability, diameter and response to vasodilator stimulation are not affected after pressure increments; yet, a prolonged lack of capillary response to the vasoconstrictor Endothelin-1 (Et-1) is observed. Considering that Et-1 is a major component of the endogenous control of retinal blood flow the present data lead to the hypothesis that prolonged or repeated IOP elevation could induce capillary disregulation contributing to neuronal damage over time.

The genesis of retinal architecture: an emerging role for mechanical interactions?

Patterns in nature have always fascinated human beings. They convey the idea of order, organization and optimization, and, to the enquiring mind, the alluring promise that understanding their building rules may uncover the forces that shaped them. In the retina, two patterns are outstanding: the stacking of cells in layers and, within the layers, the prevalent arrangement of neurons of the same type in orderly arrays, often referred to as mosaics for the crystalline-like order that some can display. Layers and mosaics have been essential keys to our present understanding of retinal circuital organization and function. Now, they may also be a precious guide in our exploration of how the retina is built. Here, we will review studies addressing the mechanisms controlling the formation of retinal mosaics and layers, illustrating common themes and unsolved problems. Among the intricacies of the building process, a world of physical forces is making its appearance. Cells are extremely complex to model as "physical entities", and many aspects of cell mechanotransduction are still obscure. Yet, recent experiments, focusing on the mechanical aspects of growth and differentiation, suggest that adopting this viewpoint will open new ways of understanding retinal formation and novel possibilities to approach retinal pathologies and repair.

Stakeholder cooperation to overcome challenges in orphan medicine development: the example of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a rare, progressive, muscle-wasting disease leading to severe disability and premature death. Treatment is currently symptomatic, but several experimental therapies are in development. Implemented care standards, validated outcome measures correlating with clinical benefit, and comprehensive information about the natural history of the disease are essential for regulatory approval of any treatment. However, for Duchenne muscular dystrophy and other rare diseases, these requirements are not always in place when potential therapies enter the clinical trial phase. A cooperative effort of stakeholders in Duchenne muscular dystrophy-including representatives from patients' groups, academia, industry, and regulatory agencies-is aimed at addressing this shortfall by identifying strategies to overcome challenges, developing the tools needed, and collecting relevant data. An open and constructive dialogue among European stakeholders has positively affected development of treatments for Duchenne muscular dystrophy; this approach could serve as a paradigm for development of treatments for rare diseases in general.

The EuroBioBank Network: 10 years of hands-on experience of collaborative, transnational biobanking for rare diseases.

The EuroBioBank (EBB) network (www.eurobiobank.org) is the first operating network of biobanks in Europe to provide human DNA, cell and tissue samples as a service to the scientific community conducting research on rare diseases (RDs). The EBB was established in 2001 to facilitate access to RD biospecimens and associated data; it obtained funding from the European Commission in 2002 (5th framework programme) and started operation in 2003. The set-up phase, during the EC funding period 2003-2006, established the basis for running the network; the following consolidation phase has seen the growth of the network through the joining of new partners, better network cohesion, improved coordination of activities, and the development of a quality-control system. During this phase the network participated in the EC-funded TREAT-NMD programme and was involved in planning of the European Biobanking and Biomolecular Resources Research Infrastructure. Recently, EBB became a partner of RD-Connect, an FP7 EU programme aimed at linking RD biobanks, registries, and bioinformatics data. Within RD-Connect, EBB contributes expertise, promotes high professional standards, and best practices in RD biobanking, is implementing integration with RD patient registries and 'omics' data, thus challenging the fragmentation of international cooperation on the field.

Dbx1-expressing cells are necessary for the survival of the mammalian anterior neural and craniofacial structures.

Development of the vertebrate forebrain and craniofacial structures are intimately linked processes, the coordinated growth of these tissues being required to ensure normal head formation. In this study, we identify five small subsets of progenitors expressing the transcription factor dbx1 in the cephalic region of developing mouse embryos at E8.5. Using genetic tracing we show that dbx1-expressing cells and their progeny have a modest contribution to the forebrain and face tissues. However, their genetic ablation triggers extensive and non cell-autonomous apoptosis as well as a decrease in proliferation in surrounding tissues, resulting in the progressive loss of most of the forebrain and frontonasal structures. Targeted ablation of the different subsets reveals that the very first dbx1-expressing progenitors are critically required for the survival of anterior neural tissues, the production and/or migration of cephalic neural crest cells and, ultimately, forebrain formation. In addition, we find that the other subsets, generated at slightly later stages, each play a specific function during head development and that their coordinated activity is required for accurate craniofacial morphogenesis. Our results demonstrate that dbx1-expressing cells have a unique function during head development, notably by controlling cell survival in a non cell-autonomous manner.

Dorsal versus ventral scales and the dorsoventral patterning of chick foot epidermis.

The dorsal and ventral scales of the chick foot can be distinguished morphologically and molecularly: the dorsal oblong overlapping scuta expressing both alpha and beta keratins, and the ventral roundish nonprotruding reticula expressing only alpha keratins. The question arises how En-1 and Lmx1, whose role in dorsoventral limb patterning has been well established, can affect skin morphogenesis, which occurs 8 to 12 days later. Forced expression of En-1 or of Lmx1 in the hindlimb have, respectively, as expected, a ventralizing or a dorsalizing effect on skin, leading to the formation of either reticula-type or scuta-type scales on both faces. In both cases, however, the scales are abnormal and even glabrous skin without any scales at all may form. The normal inductive interactions between dermis and epidermis are disturbed after En-1 or Lmx1 misexpression. Effectively, while Lmx1 endows the dermal precursors of the ventral region with scuta inducing ability, En-1 blocks the competence of the dorsal epidermis to build scuta.