Next generation sequencing (NGS) technologies have revolutionized the fields of research and diagnostics over the past decade. The possibility to routinely sequence genomes of more and more patients has provided an invaluable opportunity to collect and cross-reference data and ultimately to identify genes responsible for a given genetic disorder. These technologies have been instrumental to scientists within the NeMus department for the discovery of several genes and gene mutations involved in neuromuscular diseases.
However, analysis of the growing amounts of data generated by NGS and their relationship to complex clinical profiles requires a substantial effort in developing appropriate bioinformatics systems and tools. The "bioinformatics and Genetics" team led by Pr Christophe Béroud has developed various reference systems to address all steps of translational research. They include the most efficient pathogenicity prediction systems (UMD-Predictor and HSF coupled to the VarAFT annotation and filtration system to rapidly identify disease-causing mutations) among the many harmless genetic variations normally found in the genomes of different individuals . Thanks to this expertise, they are leading the clinical bioinformatics aspects of the RD-Connect FP7 European Project and are setting-up the French databases for Copy Number Variations [BAnque Nationale des CNV COnstitutionnels - BANCCO] identified in rare diseases and the French Rare Disease Variant Database (RDVD) collecting all variations identified in NGS experiments [http://rdvd.fr]. In addition, they have established mutation databases for genes involved in cancers, such as the BRCA1/2 genes responsible for breast/ovarian cancers and genetic diseases including NMDs, as well as patient registries, which compile genetic and clinical information for a given disease. For example, the team has developed the TREAT-NMD global registries and a national observatory for patients suffering from Facial Scapulo Humeral Dystrophy (FSHD), a neuromuscular disorder that affects primarily the muscles of the face, shoulders and upper arms. This registry contains today data from over 450 patients, making it the largest FSHD database worldwide.
Understanding the clinical data and the genetics of FSHD has been fundamental in the study of the molecular mechanisms that are defective in this disease, a field that has interested the laboratory of Dr Frédérique Magdinier for several years.
The vast majority of patients with FSHD (95%) carry a genetic mutation on chromosome 4 (at position 4q35). It is located very close to the chromosome end, or telomere, in what is referred to as the subtelomeric region. This region contains a D4Z4-repeat, a stretch of DNA in which a basic unit called D4Z4 is repeated multiple times in tandem. Healthy individuals typically display 11 to 150 D4Z4 units, while FSHD patients display a contraction of the repeat and carry only 1 to 10 D4Z4 units. But this genetic modification is different from those affecting classical genetic diseases, where a mutation is normally found within a gene and alters the gene product in a manner that compromises its function. Instead, contraction of the D4Z4-repeat locally interferes with chromatin, the structure responsible for DNA packaging and compaction inside the nucleus of a cell. Different levels of chromatin compaction are possible, which impact on local accessibility to the information encoded by the DNA: loosely compacted chromatin is permissive of gene expression, while tightly compacted chromatin is not. In healthy individuals, the D4Z4-repeat is associated with tight chromatin and inactive gene expression, but interestingly, both of these characteristics are lost in FSHD patients.
A few years ago, scientists in the laboratory of Dr Frédérique Magdinier revealed that this is due to the ability of the short D4Z4-repeats to interact with CTCF, a protein known to regulate chromatin structure. In addition, the group has shown that shortening of the repeat causes its relocalisation to the nuclear periphery, a nuclear subdomain involved in the regulation of chromatin.
From a clinical point of view, the first signs of FSHD usually appear during the second decade of life. To understand how healthy muscles all of a sudden become affected in FSHD patients, Frédérique Magdinier and her colleagues explored the molecular features of presymptomatic muscles during development.
“We found that foetal muscle displayed defects reminiscent of FSHD adult patient cells ”, explains Dr Frédérique Magdinier. “In addition, we also observed a global dysregulation of genes involved in muscle development. These results revealed that even though the symptoms of FSHD only appear as the patients reach adulthood, defects are actually already present during muscle development in foetal life”.
In addition, the team has explored large cohorts of patients with typical signs of FSHD as well as asymptomatic carriers and showed that DNA methylation, one of the main epigenetic change associated with the disease is highly variable among individuals. The team has also identified complex chromosomal rearrangements in a subset of patients highlighting the complexity of the disease locus.
These findings have established the importance of studying epigenetic changes and chromatin conformation during muscle development to understand the mechanisms leading to FSHD. Future research is aimed towards a better understanding on both the differentiation of muscle cells and the molecular mechanisms that link the organization of the 4q35 locus to the disease, through the development of cellular models such as induced Pluripotent Stem cells (iPS).
Dysferlinopathies are a group of heterogenous neuromuscular disorders that display highly variable symptoms, ranging from mild to severe functional disabilities. They are all linked to mutations in dysferlin (DYSF), a gene that encodes a large protein important upon damage for repairing the membrane that covers the muscle fibres. To date, over 400 different mutations within the DYSF gene are known to cause disease. Scientists from the MMG have a long experience in diagnosing dysferlinopathies, and have largely contributed to identifying these mutations. The laboratory of Dr Marc Bartoli has recently built a public database that compiles all the mutations found in patients and their relatives, together with the clinical information available for each individual. This database represents a valuable tool for clinicians and scientists for the understanding of this group of disorders.
Beyond their interest in diagnostics, Marc Bartoli and his colleagues have set out to explore therapeutic strategies for the treatment of dysferlinopathies. They have channelled their efforts towards developing a method based on exon-skipping, which takes advantage of the very structure of genes and the way in which they are processed for expression.
To skip or not to skip an exon – how does it work?
Structurally, eukaryotic genes are composed of exons and introns. Exons are the elements that are ultimately expressed and translated into a protein, while introns are non-coding sequences located between exons that are removed before producing the protein. Gene expression starts by transcription, a process in which the information on the DNA is copied into a molecule of another nucleic acid called RNA. Through a mechanism called splicing, the introns are removed from the RNA and the exons are joined together end-to-end. The resulting mature RNA molecule is then translated into the final protein.
Exon-skipping for the treatment of genetic disorders is based on RNA splicing. The idea is to trick the cells into splicing out the exon that carries a genetic mutation such that it is no longer translated. The resulting protein is shorter and may not be fully functional, but in some cases it retains a certain degree of functionality that can at least partially rescue the defects caused by the mutated protein. This method has proven promising for the treatment of another severe genetic disorder characterized by progressive muscle degeneration: Duchenne Muscular Dystrophy (DMD).
So, can exon-skipping be used for the treatment of dysferlinopathies?
Deletion of exon 32 had been found in an individual to cause a very mild version of the disease. The scientists therefore reasoned that a protein lacking the information from exon 32 would be sufficiently active to rescue more severe cases of dysferlinopathies.
“We developed a method for skipping this exon and asked if the resulting protein was able to rescue the defects seen in cells derived from two patients. Surprisingly, we found that although the new protein was expressed at low levels, the cells had become fully capable of repairing lesions in their membrane, indicating that the therapy had worked”, declares Dr Marc Bartoli.
These results established exon-skipping as a promising therapeutic strategy for the treatment of dysferlinopathies. Further pre-clinical studies in mice are currently on the way before translating these results into the development of clinical trials. Finally, similar approaches are also being developed for the treatment of other neuromuscular disorders.