Somatic mutations explain missing risk of multiple sclerosis.

Somatic mutations explain missing risk in multiple sclerosis

An Goris, Laboratory for Neuroimmunology, KU Leuven

1. State of the art

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, affecting 2.5 million

people worldwide and leading to important physical and cognitive disability with typical onset during

young adulthood. A genetic component has long been demonstrated and through genome-wide

association studies (GWAS) we have now identified 110 genetic risk factors1,2. Each of these is

relatively common in the population (>5%) and has a modest effect on disease risk (odds ratio of

<1.3). Currently known risk factors indicate a primary role for the immune system in MS1,2, and have

pinpointed key molecules and pathways that are targets for treatment3.

Despite the successes of GWAS, established MS risk factors explain only 27% of the variance in MS

risk between individuals2. MS patients have on average a higher load of common genetic risk factors

than healthy controls, but there is a large overlap. Hence, the key question is: what triggers whether

an individual with a certain load of common genetic risk factors does or does not get MS? Many

more common variants with even smaller effect sizes, beyond the detection limit of GWAS, exist but

are not able to explain >50% of variance in MS risk4. Novel categories of inherited genetic variants

such as rare (<1-5%) and copy number variants are being investigated but first results have been

disappointing5-7. Methodological difficulties have hampered identification of environmental risk

factors and change is not in sight.

In this project, we propose a novel explanation for the unexplained variance in MS risk between

individuals: mutations that are not inherited from the parents but have arisen newly in a subset of

cells of the patient (somatic mutations). The pathological role of somatic mutations has long been

recognized in cancer and is increasingly being uncovered in other diseases, including autoimmune

and autoinflammatory disorders8-13. In some immune-related diseases, the variable penetrance seen

within families and long attributed to “stochastic factors” can now be explained as the occurrence of

somatic mutation as “second hit” or additional factor on top of a background of inherited risk12. In

parallel, we propose that the “stochastic factors” long invoked to explain the missing part of variance

in MS risk but having escaped characterization so far14 may be explained in part by somatic

mutations. Established genetic and environmental risk factors for MS increase somatic

hypermutation rates15-17, and regulate immune cell proliferation. In particular, several risk genes

control B cell proliferation and are prone to somatic mutations in B cell proliferative diseases1,2. The

investigation of somatic mutations in MS is an entirely novel concept but is timely, given the debate

on unexplained variance in MS risk, the context where GWAS have reached their limits and the

emergence of affordable and appropriate technologies (next-generation sequencing or NGS).

2. Objectives

Overall hypothesis: Mutations that are not inherited from the parents but have newly arisen in a

subset of cells (B cells) of an individual provide a proliferation advantage to these cells. These

somatic mutations form an additional trigger, on top of a background of a high, but in itself

insufficient load of inherited risk variants, that leads to development of MS. These somatic mutations

may me detectable in either or both of the connected compartments (peripheral blood and

cerebrospinal fluid) in which B cell proliferation in MS plays a crucial role.

Aim 1. Workflow

• Establishment of workflow for intra-individual comparison of NGS data allowing detection of

somatic mosaicism enriched in B cells compared to other cells, both from the peripheral

blood and the cerebrospinal fluid (starting from strategies applied in cancer field).

Aim 2. Somatic mutations in the peripheral blood

• Do somatic mutations in B cells from the peripheral blood occur in MS at detectable rates?

CREA 2014 - An Goris - Summary p. 2

• Study of paired samples (B cells - non-B cells) from the peripheral blood of MS patients with a

high inherited risk of MS (selection of 20 from a potential of 116 patients, remaining samples

for follow-up).

Aim 3. Somatic mutations in the cerebrospinal fluid

• Do somatic mutations in B cells from the cerebrospinal fluid occur in MS at detectable rates?

• Study of paired samples (B cells - non-B cells) from the CSF of newly diagnosed MS patients

(selection of 20 from a potential of 50 patients per year, remaining samples for follow-up).

Aim 4. From mutations to pathways

• Are mutations enriched for certain pathways?

• Do key genes/pathways exist harboring newly arisen mutations in several patients or

harboring different types of MS-related variation (somatic mutations, rare inherited risk

variants, common inherited risk variants)?

3. Methodology

1. Selection of individuals and samples for sequencing

Patients are recruited through collaboration with Prof. B. Dubois (UZ Leuven). B cells are purified

using magnetic beads. We include a total of 40 samples, 20 from peripheral blood and 20 from the

cerebrospinal fluid. We select patients with a high genetic burden (based on currently known risk

factors or family history) to identify somatic mutations that act on top of an existing high inherited

risk and can explain why the threshold for clinical development of disease is crossed for this

individual and not for others with similar high risk.

2. Exome sequencing

Exome sequencing at high coverage is performed at the Genomics Core Facility of the KU Leuven,

with whom we have collaborated successfully already.

3. Bio-informatics analysis

The essence of the analysis is a search for “incompatible genotype calls” between paired samples (B

cells versus non-B cells). We apply dedicated software such as SomaticSniper or DeNovoGear 18-20.

Current algorithms allow detection of somatic variants with a frequency of >10-20% in target cells

(here B cells) and <5% in reference cells.

4. Confirmation of identified variants

Confirmation is essential to distinguish between next-generation artefacts and true mutations. This is

done by cloning followed by Sanger sequencing, hence allowing confirmation of somatic mosaicism.

5. Pathway analysis

The Ingenuity software allows testing for enrichment of specific Gene Ontology pathways. For

overlap with other types of risk variants, we use our previous existing data, as well as ongoing data

from our own group and from our privileged position as member of the International Multiple

Sclerosis Genetics Consortium (IMSGC).

6. Expected outcome

A positive outcome for aims 1-3 (‘proof of principle’ of detecting somatic mutations in MS) will result

in the confirmation of an entirely novel concept explaining part of the missing variance in MS risk.

This will open up new research lines (larger-scale investigation, follow-up) through regular funding

opportunities. First indications from aim 4 (enriched pathways, key genes harboring multiple risk

variants) will translate in personalized medicine (e.g. choice of B cell depleting therapies for patients

with dysregulated B cell proliferation) and novel, rational targets for treatments.