Autoimmune disorders of the nervous system develop as the body´s own immune cells react towards nerve tissue. The most common of these diseases is multiple sclerosis (MS), other representatives of this disease group include optic neuritis (which often evolves into MS), neuromyelitis optica, narcolepsy, neurosarcoidosis, myasthenia gravis, myelitis, polyradiculitis, polyneuritis (autoimmunity against peripheral nerves) and autoimmune encephalitides. The causes of these disorders are still poorly known, thus there are no treatments based on the underlying disease mechanism.

In relapsing form of MS, which constitute about 90% of cases, there is evidence based on genetics, environmental risk factors and treatment paradigms that leukocyte dysfunction plays a major role in the disease. However, the cells that drive autoimmune response in MS as well as in other CNS autoimmune diseases have remained unclear. Consequently, therapies are non-specific and targeted to large populations of cells with many adverse effects.

Our genetic studies in MS, in collaboration with Leena Peltonen and Janna Saarela groups, have characterized a geographical high-risk cluster in Finland with genetic founder effect (e.g. Pihlaja et al 2003, Tienari et al 2004, Kallio et al 2009, Jakkula et al 2010, IMSGC 2011) and identified about a dozen genetic risk factors of MS in Finland (demonstrating relative genetic homogeneity since the total number of risk loci is > 150 in populations world-wide). Most of the genetic risk variants confer a relatively small overall effect on MS risk, however, collectively the genetic data indicate that MS is likely a leukocyte driven disease. Even myelin basic protein (MBP), a known structural protein in myelin and a genetic risk factor of MS (Pihlaja et al 2003), has been shown to function in lymphocytes as a T cell proliferation inhibitor (Feng et al Immunity 2006) and has been identified as a risk factor of both clilhood leukemia and non-Hodkin lymphoma (Han et al Hum Immunol 2010, Hu et al Environ Mol Mutagen 2013).

In 2013 we obtained the ethical committee's approval to study blood and CSF cells with genome-wide approaches in patients with MS and other immune-mediated disorders of the nervous system. We aim to study the possible role of somatic mutations in MS. Somatic mutations are acquired mutations in DNA that occur at a low frequency by chance and can be caused by environmental factors such as viruses, chemicals and ionizing radiation. Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. There are only few studies that have studied somatic mutations in autoimmune disease (other than somatic hypermutation of immunoglobulin genes). Next generation sequencing provides single cell reads enabling the detection of rare somatic mutations and tackle with the so called “rare cell problem” in autoimmunity. To facilitate the detection of somatic mutations, we have separated the blood leukocytes into CD4+, CD8+, CD19+ and CD4-/CD8-/CD19- subpopulations.

In an explorative study targeting 986 immune-related genes with sequencing depths of >600x, we found nonsynonymous somatic mutations in 60% of the studied patients (Valori et al 2017). The mutations were enriched in CD8+ cells (85% of mutations) and 96% of the mutations persisted in follow-up. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKFZF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPA1 and STAT3). Whether this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for further studies.

We performed a genome-wide association study (GWAS) of Finnish ALS in a material composed of 500 patients (20% familial). As a control we used the Vantaa85+ material (n=515), which is a population based sample of elderly Finns. Two major loci were identified at genome-wide significant level, one on chromosome 9p21, the other on chromosome 21, representing SOD1 and the known recessive mutation SOD1*D90A (Laaksovirta et al 2010).

After more than two years of extensive next generation sequencing in a multinational collaborative effort, the chromosome 9 mutation was identified as a ggggcc-hexanucleotide repeat expansion in intron-1 of the C9ORF72 gene (Renton et al Neuron 2011, DeJesus-Hernandez et al Neuron 2011). The same mutation was found also in frontotemporal dementia FTD patients. The mutation was difficult to detect since the expansion cannot be directly sequenced. A repeat-primed PCR assay was developed, and the C9ORF72 expansion was found to be the most common mutation in Caucasian ALS, and is especially common in Finland (Majounie et al 2012). Inter-population haplotype analyses indicated a strong founder effect (Mok et al 2012). The so called “Finnish haplotype” has been found in all carriers of the expansion thus far.

Already in 2010, before finding the C9ORF72 mutation, we took skin biopsies of two patients with the risk haplotype (the last two alive patients among the 497 cases in the GWAS). In 2010-2011 these fibroblasts were de-differentiated into induced pluripotent stem (iPS) cells in collaboration with Timo Otonkoski’s group at the Biomedicum Stem Cell Center. In collaboration with Jeff Rothstein and Rita Sattler (Johns Hopkins Univ, USA) iPS cells from the two Finnish and one American ALS patient were differentiated into motoneurons for in vitro modelling of C9ORF72 ALS (C9ALS). The expansion was retained during reprogramming and culturing the cells, the cultured iPS-neurons expressed neurotransmitter receptors and were electrically active. These C9ALS iPS-neurons provided important new information on the pathogenetic mechanisms of the C9ORF72 intronic ggggcc-repeat expansion.

A large paper was published delineating important components of C9ORF72 expansion pathophysiology (Donnelly et al Neuron 2013): (i) C9ALS iPS-neurons were abnormally sensitive to glutamate toxicity. (ii) The expansion formed intranuclear RNA inclusions. (iii) By using proteome arrays against the ggggcc-repeat, protein:RNA co-immunoprecipitation, and histochemistry the ADARB2 protein was shown to be one of the proteins that bind to the ggggcc-repeat. (ADARB2 is an enzyme that edits glutamate receptor mRNA and thereby regulates the receptors activity). (iv) Down-regulation of ADARB2 by siRNA increased wild-type iPS-neuron’s sensitivity to glutamate. (v) Antisense oligonucleotide therapy against the ggggcc-repeat expansion was able to partially rescue the toxicity of the expansion as evidenced by the reduced number of RNA inclusions and better glutamate tolerance of the C9ALS iPS-neurons.  These results indicate that the RNA generated by the expansion exhibits an abnormal tendency to bind nuclear proteins and render motor neurons sensitive to glutamate. Therapy with antisense oligonucleotide or small molecules inhibiting RNA-protein interaction are theoretically possible.

Vantaa85+ study was initiated by prof Raimo Sulkava (University of Kuopio, Kuopio, Finland) and Dr. Leena Niinistö (Katriina Hospital, Vantaa, Finland).  The study population includes all persons aged 85 years or over who were living in the city of Vantaa, on April 1, 1991. Of the 601 eligible subjects, peripheral blood (and DNA) samples have been obtained from 517 study subjects, and of these 306 have been neuropathologically examined. Vantaa 85+ is worldwide the only population-based study with over 50% autopsy rate and neuropathological examination of subjects.  Several common age-related brain pathologies have been analysed in the neuropathological subsample. These include Alzheimer pathologies [beta-amyloid pathology and neurofibrillary tau pathology], cerebrovascular amyloid angiopathy, brainstem and neocortical Lewy-related pathology (cytoplasmic Lewy bodies and Lewy neurites), brain infarcts and brain haemorrhages. We have recently shown that Alzheimer’s type of tau-pathology is the strongest single contributor of dementia in this “oldest old” population (Tanskanen et al 2017). We also found that multiple pathologies occurring simultaneously increase the risk of dementia substantially (Tanskanen et al 2017).

We found the A673T variant of the APP gene that reportedly protects against Alzheimer’s disease in one subject (0.2%) of the Vantaa85+ study. She lived till age 104.8 years (second highest age-at-death in the cohort) and neuropathological analysis showed very little beta-amyloid pathology (Cerad score 0). The low amount of parenchymal beta-amyloid pathology at the age of 104.8 yrs supports the concept that the A673T variant protects the brain against beta-amyloid pathology (Kero et al 2013).

Vantaa85+ was the control group in the ALS-GWAS. The GWAS data has enabled genome-wide analysis of genetic risk factors of many age-related neuropathologies. APOE ε4 is currently the strongest known risk factor for Alzheimer’s disease. In the Vantaa85+ material we have found strong associations of APOE ε4 with the cortical beta-amyloid (p=4.91x10-17)  cerebrovascular amyloid (p=9.87x10-11) and neurofibrillary tau-pathology (p=3.45x10-7) indicating that APOE ε4 is a major risk factor of all neuropathological features of AD (Peuralinna et al 2011).  In the Vantaa85+ study we demonstrated the first evidence for a role of genetic variation in the alpha-synuclein (SNCA) selectively in tau pathology (Peuralinna et al 2007).

The genetic analysis of neocortical Lewy-related pathology has yielded interesting novel results. Dementia with Lewy bodies is an alpha-synucleinopathy characterized by the neocortical Lewy-related pathology and, although relatively common neurodegenerative disorder, its genetic background is still poorly understood. In Vantaa85+ material 15% of the subjects had neocortical Lewy-related pathology. In the GWAS we detected one genome-wide significant signal within the HLA complex on chromosome 6p21 (close to HLA-DPB1 gene) and 5 loci with suggestive allelic associations (p<10-5 on chromosomes 15q14, 2p21, 2q31, 18p11 and 5q23). The chromosome 2p21 locus, which codes for beta-spectrin (SPTBN1), a component of Lewy bodies, was replicated in a British Medical Research Council Cognitive Function and Ageing Study (CFAS) material (Peuralinna et al 2015). APOE e4 associated with neocortical Lewy-related pathology only in those subjects with concomitant Alzheimer’s pathology and these subjects probably represent the so called Lewy-body variant of Alzheimer’s disease.