Using Next Generation Sequencing to Unravel the Pathogenesis of Sporadic Inclusion Body Myositis - The International IBM Consortium Genetic Study

The commonest muscle disease that occurs in patients over the age of 45 years is a muscle wasting disease called inclusion body myositis (IBM). Patients typically develop progressive muscle wasting and weakness that progresses and causes marked disability and ultimately death from immobility over the course of around 10 years. There are no effective treatment for patients with IBM. The precise cause of this muscle disease is not known. However, on muscle biopsies from patients there seems to be a combination of some mild inflammation in the muscle and also an accumulation of abnormal proteins, similar to the accumulated proteins that are seen in the brains of patients with neurodegenerative diseases such as Alzheimer's, fronto-temporal dementia and motor neurone disease.

Previous research has indicated that there may be genetic factors that predispose people to getting IBM but the previous studies have been quite small and not conclusive. In this research we have brought together experts in IBM from all over the world including Europe, USA and Australia to generate increased awareness of IBM, define diagnostic criteria, collect clinical information and DNA. Over the last three years we have been able to collect the largest group ever of IBM patients and DNA samples - approximately 950 cases and this number will be over 1000 once this study begins. The patient DNA and muscle tissue has been carefully stored for this work. This very large collection of DNA has put us in a very good position to undertake much more detailed genetic studies than have ever been done before to try and work out what the genetic risks factors and genes are that predispose people to this devastating disease.

We plan to use the latest next generation sequencing techniques to unravel all the coding variants (those that alter proteins) that are present in 200 IBM patients DNA samples in comparison with 200 patients that are controls with normal muscles. We will analyze the DNA that we have already extracted from patients muscle tissue as this is the best diagnostic group. We will replicate the variants found in a further 700 IBM cases and over 2200 other controls. We are highly experienced in next generation sequencing technology and this has been strengthened by the recent award of a Wellcome Trust equipment grant to purchase the latest next generation sequencer. Recently we have used these techniques to identify the genetic causes of other neuromuscular disorders.

In comparison with other disorders like Alzheimer's disease, where proteins are aggregated in the brain as opposed to the muscle as in IBM, the greatest advancement have been made with the identification of disease genes and genetic risk factors. If we can work out what the key genes are and how these disease causing pathways function, we will pave the way for new therapies and treatments to help patients.

1. Library preparation for exome sequencing
In each case high quality DNA (A260/280 ratio >1.8) has been extracted. We have switched over to the Illumina TruSeq Enrichment Kit as it allows six samples to be pooled for exome sequencing and is therefore far more cost effective. The enrichment method is a standard protocol from Illumina starting with the TruSeq sample preparation, pooling of six samples, capture of targeted regions with the TruSeq exome kit and then sequencing. The HiSeq has a very large capacity for sequencing, with the TruSeq enrichment our current most cost effective run is 100bp paired-end reads which gives 60 million reads per sample, at around 68 fold coverage (2 samples per lane). Depending on cost we can modify the runs in several ways, usually by extending read length and changing samples per lane.

2. Exome sequence data processing and variant calling
After the genome analyser generates the raw sequencing images (image analysis) there are several steps involved in the read conversion to variant calling. These include initial base calling, sequence alignment, building, indexing, sorting SAM files, recalibrating quality scores, removing duplicates and merging lanes. Variants are called and filtered based on quality control (read depth, quality scores etc). A genotype file is produced and then annotation and prediction of consequence (amino-acid change, predicted damage of variant) is carried out using the package Sorting Intolerant From Tolerant (SIFT).

3. Statistical analysis
This will primarily focus on gene level comparisons testing a reduced set of genes identified as harbouring non-synonymous coding variants or splice variants that may contribute to risk of IBM. Exome sequence data will be filtered first to remove common variants found in publicly available data from dbSNP and the 1000 Genomes Project. A second level of filtering will be used to extract non-synonymous coding variants and splice variants using existing exome annota

Who will benefit
Importance and unmet health need for patients and the economy: Inclusion body myositis is the most common acquired muscle disease in patients over the age of forty years. It causes progressive major disability and often premature death after 5-10 years from the consequences of immobility. It represents a significant unmet health need in the UK-worldwide. Since onset is often in prime working life and patients often become significantly disabled quickly and cannot contribute to the economy. A systematic genetic study using the latest genetic technology has not been undertaken. Limited studies have suggested a HLA association, however the disease is unresponsive to immunosuppression and discovery of new mechanisms pathways is required. Key beneficiaries from this research include 1. The immediate and wider science communities, 2. Key established and new industry partners who can develop new paradigms for drug testing based on new genetic discovery 3. Patient organisations and patients. Until now IBM patients have been a relatively neglected group experiencing delayed and misdiagnosis. Professor Hanna and the MRC Centre for translational research have existing partnerships with the major patient organisations linked to IBM namely the myositis support group where Professor Hanna is an advisor helped plan annual patient meetings and the Muscular Dystrophy Campaign that also support IBM patient groups.

How will they benefit
Potential areas for exploitation from this research: Discovery of new genetic associations for IBM will have significant potential to build on existing collaborations with industry and develop new industry collaborations. Potential new areas arising from this research include- identification of new target pathways that might be druggable, the possibility of developing new or taking advantage of existing animal models and of developing new cellular models. All these possibilities will be of interest to industry especially for testing new therapies based on the genetic discovery of new targets. We have established collaborations with the Senexis (Cambridge biotech) to screen anti protein aggregate small molecules and we are currently using a nematode and a myoblast line for screening. However, Senexis- (see MRC Centre Senexis collaboration at www.cnmd.ac.uk) have access to very large number of potential agents- new genetic discovery could direct selection. We have an existing collaboration with Cytrx USA and completing a proof of principle heat shock protein upregulation experimental medicine study in IBM- this relationship will be developed by new genetic discovery. New IBM genetic associations has potential for new genetic testing paradigms for risk stratification and potential for commercialisation of genetic testing.
With Patients and Patient Organisations- the MRC Centre for Neuromuscular Diseases has established effective and regular communication channels with the key patient groups representing IBM. Results of this research along with implications and impact for patients and families will be discussed at the annual Myositis Support Group meeting, the annual MRC Centre patient group meeting and the Muscular Dystrophy Campaign patient group meetings and local branch meetings. All our latest IBM research is posted on the MRC Centre website, the MDC website and the Myositis support group newsletter. With policy makers and NHS commissioners-Professor Hanna is actively engaged with the cross party parliamentary group for neuromuscular diseases including IBM and has given evidence in parliament about the impact of neuromuscular diseases including IBM and has worked with this group and the commissioners to develop standards of care for patients- this work is ongoing and can include implications of this genetic research discovery for patient care eg genetic risk assessment.