Overview of ALS Genetics  Parts of what’s known and a glimpse of what’s next…

1. Overview of ALS GeneticsParts of what’s known and a glimpse of what’s next… Patrick Dion, Ph.D. Neurology and Neurosurgery McGill University

2. Human Genome Basics • 3 billions base pairs. • Contains protein-coding and noncoding DNA. • 19,000-20,000 protein-coding genes (2% of the genome). • Exomes refers to the 2% coding DNA share. • Noncoding is associated with regulation of expression, chromosome architecture and epigenetic regulation. • First sequencing draft completed in 2001. • Several thousands genomes have now been sequenced. • Available through various databases (e.g. 1kGP, ExaC, EVS). • Regardless of race we are 99.9% identical at the genome level.

3. Human Genome Basics • Distinctive markers exist all across the genome. • [Large] RFLP, tandem repeat, Copy Number Variants (CNVs) • [Small] Single nucleotide polymorphisms (SNPs). • SNPs are common variants found in >1% of the population at 1,000 bp across. • When in coding regions SNPs can be synonymous or nonsynonymous. • Distinction between SNPs and single nucleotide variants (SNVs).

4. Discovery Approaches • Classical linkage analysis • Candidate genes association • Genome Wide Association Study (GWAS) • Use of common variants to identify regions shared by affected. • Whole Exome Sequencing (WES) • Seeks to filter rare coding variants to identify to identify deleterious ones. • No need for controls except when population specific. • Whole Genome Sequencing (WGS) • Seeks to filter all variants (coding and not coding) • WES and WGS data can also be used to conduct association studies using common variants and rare ones (e.g. Minor Allele Frequency < 1%)

5. Variants filtering of WES and WGS

6. FALS and SALS • Overall ALS is the most common rare disease (2/100,00). • FALS (familial) • 5-10% of cases of ALS • Primarily autosomal dominant (AD) segregation of definite, probable or possible individuals. • SALS(sporadic) • No family history • Clinically indistinguishable • Except age of onset and sex distribution • This classification should not overshadow the fact that FALS and SALS DO share common genetic causes. • Moreover environmental/stochastic factors can affect genetically susceptible individuals.

7. Just how many ALS genes are there? • http://alsod.iop.kcl.ac.uk/home.aspx • Updated once a year. • Between the discovery of the first causative gene (SOD1) in 1993 and now,126 genes were “linked” to ALS. • [+] Classify the genes according to phenotype, geographical distribution and method of identification. • [-] Includes > 50% of genes for which associations are either weak and/or were never replicated in independent studies. • Nonetheless a valuable and objective online to use assessment tool to consult about the causal nature of specific variants. • The risk linked to ALS genes can be classified according to multiple factors.

8. High Risk Genes

9. Superoxide Dismutase-1 SOD1 [ALS1] • Originally found using Classical linkage analysis and FALS. • Accounts for ~20% of FALS forms (2% of SALS). • >160 mutations identified all over SOD1. • All dominant except for D90A and D96N, recessive in some cases • Some mutations affect survival time: • A4V rapid progression D90A slow progression • Some affect disease onset: • G37R with early onset • Most mutations trigger a shift of the folding equilibrium toward poorly structured SOD monomers. • A great number of mechanisms are proposed to be involved, however, distinguishing cause from effect and identifying the critical processes remains challenging

10. TAR DNA Binding ProteinTARDBP (TDP-43) [ALS10] • [Candidate gene approach] • In 2008 the gene was screened for mutations as its product was a prominent product of ubiquitinated cytoplasmic inclusions in the CNS tissues of FTD and ALS. • Accounts for ~4% of FALS forms (<1 % of SALS). • > 47 Missense and one truncating variants. • All variants are dominants. • Pathogenic variants are mostly in the C-terminus which is involved in RNA binding and splicing.

11. Fused in SarcomaFUS[ALS6] • [Candidate gene approach and locus approaches] (2009) • Accounts for ~4% of FALS forms (<1 % of SALS). • Autosomal dominant and recessive. • Age of onset younger (< 40yrs with cases during teens). • Faster progression than TARDBP and SOD1 cases • > 50 Missense and one truncating variants. • All variants are dominants. • Pathogenic variants are mostly in the C-terminus.

12. Chromosome 9 open reading frame C9orf72[ALS-FTD] • [GWAS and locus approaches] (2011) • Accounts for ~40% of European descent familial ALS-FTD cases (10 % of Asians) and 7% of SALS. • Large intronic repeat expansion (GGGGCC or G4C2). • >30 repeats pathogenic, 15-30 not very pathogenic but recently observed to be with ATXN2 intermediate CAG expansion. • Both gain and loss of functions are under consideration. • Multiple pathogenic avenues.. • RNA foci and sequestration of RNA binding components. • Non-ATG (RAN) translation of repeat derived dipeptide accumulation in the CNS (GR > PR > GA > AP > GP). • Dysregulation of its potential DENN Rab-GEFs activity on membrane trafficking. • Disrupts of nucleocytoplasmic transport of mRNA.

13. Low Risk Genes

14. Percentage of ALS genetically explained Nat Neurosci. 2014 Jan; 17(1): 17–23.

15. New genes 2015 and up * * * * *Aggregation tests termed as burden tests collapse information for multiple genetic variants into a single genetic score and test for association between this score and a trait. A simple approach summarizes genotype information by comparing the number of minor alleles (˂ 1%) across all variants in the sequencing data of multiple cases and controls.

16. Growing percentage of SALS genetically explained 17 % Nat Neurosci. 2014 Jan; 17(1): 17–23. Neurology 2017;89:226-233

17. A rapidly increasing number of ALS genes Bettencourt & Houlden, Nat Neuroscience 2015

18. Rate of ALS gene Discovery >100 ALS genes? C21ORF2 UNC13A MOBP TIA1 SCFD1 ANXA11 CCNF 2017 Rate of new gene discovery has reached a plateau despite increased sequencing efforts

19. Rate of gene discovery • “Plateau” Stage • Several candidate genes without strong evidence • Functional relevance unknown • Limit reached for current technologies • Require larger cohorts to detect small effect sizes • Deep phenotyping and extreme phenotypes • New directions: • Large consortia (MinE) • Whole genome sequencing • Rare and small structural variants • Epigenetics

20. Other themes tested for ALS Genetics • Oligogenicity • Possibility that ALS is caused by two (or more) variants concurrently that would not independently cause disease. • Studies have observed patients with multiple mutations in “causal” genes. • e.g. C9orf72 expansion with an OPTN mutation of unknown pathogenicity • Does severity of disease increase with more ALS mutations? • De novo mutations • Mutations can occur in germinal cells, not present in either parent • Sporadic ALS patients have no family history, but could pass new mutations to children? • De novo isnot commonly observed in genetic studies, not a common cause of ALS

21. Thank you! patrick.a.dion@mcgill.ca