2015 · Clinical · Scientific

ENCALS Thursday 21st May 2015 – Oral session 1 – Genes and Genomics

The ENCALS meeting this year has been held at Trinity College in Dublin. This suggestive place hosted more than 250 participants and several international guests as the eminent researcher Prof. John E. Landers,Professor of Neurology at University of Massachusetts, who opened the “Genes and Genomics” session with a talk entitled “Using Next-Generation Sequencing to Dissect Amyotrophic lateral sclerosis (ALS) Pathogenesis”. Prof. Landers pointed out how the investigation of rare variants are important in the discovery of pathways underlying the neurodegenerative mechanism of rare diseases and how exome sequencing became an important strategy for identifying human disease genes by spotting rare variants.

Using exome sequencing in two large ALS families (Wu et al. Nature 2012), Landers and collaborators discovered that different mutations in profilin 1 (PFN1) gene cause familial ALS. PFN1 is important for the conversion of monomeric (G)-actin to filamentous (F)-actin and in the cytoskeleton integrity. After this finding, Prof. Landers conducted a study involving the overexpression of the human wild type (WT) and PFN1 mutants in flies. Flies overexpressing PFN1 mutants did not seem to be affected as much as flies overexpressing WT PFN1, showing that the latter one exerted a toxic function which led to a decrease in survival. This indicated that the mutations in PFN1 found by Landers and collaborators lead to a loss of function of the PFN1 protein.
However, it is not always possible to obtain a second affected family member and causative genes cannot be identified through segregation analysis. Prof. Landers and collaborators overcame the problem performing an exome-wide rare variant analysis on 363 familial ALS (FALS) cases (Smith et al. Neuron 2014). This analysis increased the statistical power by grouping together rare variants present in the same region or gene and increasing the number of rare alleles tested at the same time. The study confirmed the involvement of the MATR3 as a causative gene in familial ALS and identified a new gene involved in microtubule networking and cytoskeleton integrity, TUBA4A, which encodes for Tubulin Alpha 4 protein. This finding was further confirmed in 272 FALS cases and 5,510 internal controls, increasing evidences of a cytoskeleton alterations contributing to ALS pathogenesis. Image analysis on individual neuron cells transfected with a mutant of TUBA4A showed an increased neurodegeneration over time.
Looking at future studies, Prof. Landers concluded his talk saying that screening of modifier genes that will use a library of cytoskeleton related RNA interference (RNAi) will possibly lead us to rescue models that express TUBA4A mutants or that overexpress PFN1.

Dr Russel McLaughlin, researcher at Trinity College Dublin, who chaired the Genes and Genomics session, was the second speaker of the session. He presented an interesting study based on the findings that Schizophrenia and suicidal behaviour were over-represented in kindreds from an ALS cohort of Irish population (Byrne et al. ANN NEUROL 2013). Dr McLaughlin further investigated the genetic overlap between ALS and Schizophrenia by estimating the genetic correlation between the two traits. This can be informative, because it tells us the percentage of common genetic factors that influence both traits. The rate of the overlap between ALS and Schizophrenia was obtained performing an LD score regression analysis, which uses summary statistics of genome wide association studies (GWAS) data and represents a powerful method that can distinguish between inflation due to confounding bias and the effect due to the sum of many small genetic effects (polygenicity). Dr McLaughlin estimated that the genetic correlation between ALS and schizophrenia was substantial (14.3%). Calculating the schizophrenia polygenic risk scores in ALS, he showed that the shared risk between the two traits was due to small genetic effects.

H. el Oussini, a PhD student of the Faculty of Medicine of Strasbourg, presented her work on “the role of Serotonin 2b Receptor preventing microglia degeneration and disease progression in ALS”. Serotonergic receptors were studied in brainstem and spinal cord of SOD1 G86R end-stage mice and the HTR2B gene, encoding for 5-HT2B receptor, was found up-regulated. A functional analysis of this receptor was conducted crossing the SOD1 G86R mutant mouse with a knockout mouse for HTR2B, evidencing a correlation between up-regulation of this receptor and survival. Mice carriers for SOD1 G86R and crossed SOD1 G86R mice knockout for HTR2B reached the disease onset at the same time, but the latter ones were characterized by a shorter survival. The 5-HT2B receptor was found up-regulated only in the microglial fraction of end-stage mice and SOD1 mutant/HTR2B knockout mice showed a fragmentation of the microglial cytoplasm, indicating a modifier effect of the receptor on the microglial morphology.
Oussini showed how tag SNP screening of HTR2B gene in a Dutch population cohort of ALS cases and matched controls did not highlight any significant difference between the two groups. However, four SNPs on HTR2B gene seemed to be correlated with survival expectancy in patients.
Furthermore, a correlation between HTR2B SNPs associated with short survival was found with a decreased 5-HT2B receptor level in spinal cord samples of seven ALS patients, finding that, Oussini told, must be confirmed in a larger cohort of samples.

Prof. Jochen H Weishaupt, consultant of the Department of Neurology at Ulm University, was the fourth speaker of the session. He illustrated the results obtained from exome sequencing of 252 familial ALS, negative for SOD1 and C9ORF72, and 827 control individuals (Freischmidt et al. Nat Neurosci 2015). Through a gene-based rare variant analysis, eight loss-of-function mutations were found in the TBK1 gene (encoding TANK-binding kinase 1) in 13 familial ALS pedigrees. None of the found mutations were observed in a screen of 1,010 sporadic ALS and 650 additional control individuals.
Prof. Weishaupt showed that in vitro experiments confirmed the loss of expression of TBK1 loss-of-function mutant alleles, resulting in an haploinsufficiency of TBK1. Moreover, the C-terminal TBK1 coiled-coil domain (CCD2) mutants resulted in a loss of interaction with the TBK1 adaptor protein optineurin, already described to be involved in ALS pathogenesis.
Patients, carriers of TBK1 loss-of-function mutations, had cognitive impairment or fronto-temporal dementia (FTD), showing a new overlap between ALS and FTD.

Beyond my role of reporting the interesting talks of the first session of the ENCALS meeting 2015, I was invited to give the last talk of the session.
As a post-doc at the Institute of Psychiatry, Psychology and Neuroscience (IoPPN) of the King’s College London, I presented a systematic review and meta-analysis of the studies published between 2010 and 2014 regarding the risk of ATXN2 intermediate CAG expansion alleles in ALS. Since the first paper appeared (Elden et al. Nature 2010), different case-control studies were carried out in several populations. However, they reported contrasting results regarding the minimum intermediate CAG repeat number that increases the risk of ALS. The meta-analysis included thirteen studies selected from the literature and two novel case-control studies. Calculating the relative risk of each intermediate allele in the range of 24 and 34 repeats, the analysis confirmed a significant increase in the risk of ALS of individuals carrying ATXN2 alleles of CAG repeat size 29 to 33. Moreover, this study showed that, rather than an effect on age of onset, as it is expected in trinucleotide expansion diseases, ATXN2 trinucleotide repeat length was proportional to the risk of ALS, showing an exponential relationship between alleles 29 and 32.

The discoveries reported in this session increase the number of genes involved in ALS and try to better define the pathogenic role of the known ones. As suggested by Prof. Landers, it is not only important to look at a single gene, but at all the known genes to try and find common pathways underlying the neurodegenerative mechanism of ALS.