Gene chips for diagnosis of NMDs - an update from the NMD-chip project

NMD-chip (www.nmd-chip.eu) is a scientific project funded by the European Union. Its aim is to design, develop and validate new sensitive high-throughput DNA microarrays ("gene chips") to diagnose patients affected by inherited neuromuscular disorders (NMDs).

The project addresses two main issues facing neuromuscular genetics: diagnosis of mutations in genes already known to be involved in NMDs and discovery of new genes not previously known to be responsible for NMDs. Even though the gene responsible for the condition can be determined in around 60 to 70% of cases, the fact that many different genes may need to be tested means that obtaining a genetic diagnosis is often a lengthy and expensive process involving testing of genes one by one until the disease-causing mutation is found. Chip technology can allow all the potentially relevant genes in a patient’s DNA to be tested simultaneously, which is a much faster and cheaper process. The project is designing chips for the diagnosis of mutations already known to cause Duchenne / Becker muscular dystrophies (DMD/BMD), limb girdle muscular dystrophies (LGMD), congenital muscular dystrophies (CMD), and hereditary motor-sensory neuropathies or Charcot-Marie-Tooth neuropathies (CMT). These diagnostic chips are described as "known-gene chips".

However, another problem facing doctors and patients is that not all genes responsible for neuromuscular disorders have yet been discovered, and an estimated 30 to 40% of patients are left without a diagnosis even once all relevant known genes have been tested. The NMD-chip project is therefore also developing "candidate-gene chips" to look for mutations in patients who have a neuromuscular disorder where the gene responsible has not yet been found.

The scientific strategy of the project is to design 4 types of chip: two for known genes, and two for candidate genes. In each case, both comparative genomic hybridization (CGH) arrays and sequence capture chips are being developed -the former to test for insertions and duplications and the latter for point mutations. The technology used, developed by Roche-Nimblegen, allows up to 2.1 million probes to be spotted on one chip, and the resulting images to be analyzed with a 2-micron resolution scanner. The project is making use of the new generation of 12-plex chips, each chamber comprising 72,000 probes covering all the exons, intron flanking regions, and when known, deep intronic mutation spots of known genes for each group of pathology (CMD / LGMD / DMD / CMT).

The project has now reached its half-way point, and the first-generation CGH chips for known genes have been validated. Two CGH-arrays containing probes covering all genes known to be involved in NMDs have been developed. The first array is dedicated to muscular dystrophies (including both dominant and recessive LGMDs), congenital muscular dystrophies, and congenital myopathies. The second array is dedicated to hereditary motor sensory neuropathies. These chips will now become part of the standard diagnostics workflow to progressively replace the current techniques.

Sequence capture chip design was initiated after validation of the CGH arrays, and the SC chips are in production and validation. Results for LGMD/DMD/CMD sequence selection will soon be compared to another capture technique "in solution" called Sure Select from Agilent, which it has been suggested may give better results. Both technologies will be evaluated, and the better one will be selected for the next steps of the project.

In the research part of the project, the development of the candidate-gene chips, the chips have been designed and are currently under validation. These chips aim to identify new genes involved in the three groups of diseases of the project, namely congenital muscular dystrophies (CMD), Charcot-Marie-Tooth diseases (CMT) and limb-girdle muscular dystrophies (LGMD). A list of the most likely candidate genes for CMD, CMT and LGMD was developed, and on the basis of this, CGH-arrays for the 3 groups of diseases have been designed.

A large reference materials database has also been created to collect relevant samples to be used as positive controls on the chips. From a bioinformatics point of view, several tools have been created to improve the chip design, to collect all the data, and to analyse the pathogenicity of the mutations detected by the chips. 

For more information visit the project's website at www.nmd-chip.eu.