Rare variants with large effects provide functional insights into the pathology of migraine subtypes, with and without aura

Migraine is a complex neurovascular disease with a range of severity and symptoms, yet mostly studied as one phenotype in genome-wide association studies (GWAS). Here we combine large GWAS datasets from six European populations to study the main migraine subtypes, migraine with aura (MA) and migraine without aura (MO). We identified four new MA-associated variants (in PRRT2, PALMD, ABO and LRRK2) and classified 13 MO-associated variants. Rare variants with large effects highlight three genes. A rare frameshift variant in brain-expressed PRRT2 confers large risk of MA and epilepsy, but not MO. A burden test of rare loss-of-function variants in SCN11A, encoding a neuron-expressed sodium channel with a key role in pain sensation, shows strong protection against migraine. Finally, a rare variant with cis-regulatory effects on KCNK5 confers large protection against migraine and brain aneurysms. Our findings offer new insights with therapeutic potential into the complex biology of migraine and its subtypes.


Introduction
Reactome is a curated database of pathways and reactions in human biology.Reactions can be considered as pathway 'steps'.Reactome defines a 'reaction' as any event in biology that changes the state of a biological molecule.Binding, activation, translocation, degradation and classical biochemical events involving a catalyst are all reactions.Information in the database is authored by expert biologists, entered and maintained by Reactome's team of curators and editorial staff.Reactome content frequently cross-references other resources e.g.NCBI, Ensembl, UniProt, KEGG (Gene and Compound), ChEBI, PubMed and GO.Orthologous reactions inferred from annotation for Homo sapiens are available for 17 non-human species including mouse, rat, chicken, puffer fish, worm, fly, yeast, rice, and Arabidopsis.Pathways are represented by simple diagrams following an SBGN-like format.
Reactome's annotated data describe reactions possible if all annotated proteins and small molecules were present and active simultaneously in a cell.By overlaying an experimental dataset on these annotations, a user can perform a pathway over-representation analysis.By overlaying quantitative expression data or time series, a user can visualize the extent of change in affected pathways and its progression.A binomial test is used to calculate the probability shown for each result, and the p-values are corrected for the multiple testing (Benjamini-Hochberg procedure) that arises from evaluating the submitted list of identifiers against every pathway.
To learn more about our Pathway Analysis, please have a look at our relevant publications: Reactome pathway analysis: a high-performance in-memory approach.BMC Bioinformatics, 18.

Properties
This is an overrepresentation analysis: A statistical (hypergeometric distribution) test that determines whether certain Reactome pathways are over-represented (enriched) in the submitted data.It answers the question 'Does my list contain more proteins for pathway X than would be expected by chance?'This test produces a probability score, which is corrected for false discovery rate using the Benjamani-Hochberg method. •

Most significant pathways
The following table shows the 25 most relevant pathways sorted by p-value.

Pathways details
For every pathway of the most significant pathways, we present its diagram, as well as a short summary, its bibliography and the list of inputs found in it.
Myogenesis, the formation of muscle tissue, is a complex process involving steps of cell proliferation mediated by growth factor signaling, cell differentiation, reorganization of cells to form myotubes, and cell fusion.Here, one regulatory feature of this process has been annotated, the signaling cascade initiated by CDO (cell-adhesion-molecule-related/downregulated by oncogenes) and associated co-receptors.CDO/Cdon is a type I transmembrane multifunctional co-receptor consisting of five immunoglobulin and three fibronectin type III (FNIII) repeats in the extracellular domain, and an intracellular domain with no identifiable motifs.It has been implicated in enhancing muscle differentiation in promyogenic cells.CDO exert its promyogenic effects as a component of multiprotein complexes that include the closely related factor Boc, the Ig superfamily receptor neogenin and its ligand netrin-3, and the adhesion molecules N-and M-cadherin.CDO modulates the Cdc42 and p38 mitogen-activated protein kinase (MAPK) pathways via a direct association with two scaffold-type proteins, JLP and Bnip-2, to regulate activities of myogenic bHLH factors and myogenic differentiation.CDO activates myogenic bHLH factors via enhanced heterodimer formation, most likely by inducing hyper-phosphorylation of E proteins.
Myogenic basic helix-loop-helix (bHLH) proteins are master regulatory proteins that activate the transcription of many muscle-specific genes during myogenesis.These myogenic bHLH proteins also referred to as MyoD family includes four members, MyoD, myogenin, myf5 and MRF4.These myogenic factors dimerize with E-proteins such as E12/E47, ITF-2 and HEB to form heterodimeric complexes that bind to a conserved DNA sequence known as the E box, which is present in the promoters and enhancers of most muscle-specific genes.Myocyte enhancer binding factor 2 (MEF2), which is a member of the MADS box family, also plays an important role in muscle differentiation.
MEF2 activates transcription by binding to the consensus sequence, called the MEF2-binding site, which is also found in the control regions of numerous muscle-specific genes.MEF2 and myogenic bHLH proteins synergistically activate expression of muscle-specific genes via protein-protein interactions between DNA-binding domains of these heterologous classes of transcription factors.
Neurotrophin functions are mediated by binding of the secreted neurotrophin homodimers to their common neurotrophin receptor p75NTR, and to their cognate tropomyosin related kinase (TRK) receptor.NGF binds to TRKA, BDNF and NT4 bind to TRKB, NT3 binds to TRKC.A tri-molecular signalling complex (NGF-p75NTR-TRKA) might also be possible.When the co-receptor sortilin is present at the cell surface, proNGF preferentially interacts with a p75NTR:sortilin complex.Thus, proNGF, which does not bind TRKA, discriminates between TRKA and p75NTR, in cells that express both receptors.The same is true for proBDNF.Pro-neurotrophin binding to p75NTR:sortilin activates an apoptotic cascade, which may be involved in cell death after injury, and in neurodegenerative diseases such as Alzheimer's dementia.
Neurotrophin-induced increase in Signal transducer and activator of transcription 3 (STAT3; acutephase response factor) activation appears to underly several downstream functions of neurotrophin signalling, such as transcription of immediate early genes, proliferation arrest, and neurite outgrowth.

Date
Action Author Perhaps the most important and widely studied blood group is the ABO blood group.It consists of antigens found on the outer surface of red cells and corresponding antibodies in plasma.The majority of the world's population (~80%) are 'secretors' which means that the antigens present in their blood will also be found in other body fluids such as saliva.An individual can be a Secretor (Se) or a non-secretor (se) and this is completely independent of whether the individual is of blood type A, B, AB, or O. From a very early age, the immune system develops antibodies against whichever ABO blood group antigens are not found on the individual's RBCs.Thus, a blood group A individual will have anti-B antibodies and a blood group B individual will have anti-A antibodies.Individuals with the most common blood group, O, will have both anti-A and anti-B in their plasma.Blood group AB is the least common, and these individuals will have neither anti-A nor anti-B in their plasma.
The primary structure of these antigens is an oligosaccharide precursor sequence on to which one or more sugars are attached at specific locations.The blood group oligosaccharide antigens A, B and H are produced by enzymes expressed by these genes and form the basis of the ABO 'blood type' phenotypes.A and B antigens were originally identified on red blood cells (RBCs) but later identified on other cell types and in bodily secretions.The ABO blood group system is important in blood transfusion, cell/tissue/organ transplantation and forensic evidence at crime scenes.
The H antigen is formed with the addition of a fucose sugar onto one of two precursor oligosaccharide sequences (Type 1 chains are Gal 1,3 GlcNAc 1,3 Gal R and Type 2 chains are Gal 1,4 GlcNAc 1,3 Gal R; where R is a glycoprotein (Type 1) or glycosphingolipid (Type 2).Type 2 chains are only found on RBCs, epithelial cells and endothelial cells.The H gene expressed in hematopoietic cells produces -1,2-fucosyltransferase 1 (FUT1) which adds a fucose to Type 2 chains to form the H antigen in non-secretors.Type 1 chains are found in secretors.The Se gene expressed in secretory glands produces -1,2-fucosyltransferase 2 (FUT2) which adds a fucose to Type 1 chains to form the H antigen in secretors.NGF processing (R-HSA-167060) 9.
All neurotrophins (NTs) are generated as pre-pro-neurotrophin precursors.The signal peptide is cleaved off as NT is associated with the endoplasmic reticulum (ER).The resulting pro-NT can form a homodimer spontaneously which then transits to the Golgi apparatus and then onto the trans-Golgi network (TGN).Resident protein convertases (PCs) can cleave off the pro-sequence and mature NT is is targeted to constitutively released vesicles.The pro-NT form can also be released to the extracellular region.Signalling to p38 via RIT and RIN (R-HSA-187706) 11.

References
RIT and RIN are two small guanine nucleotide binding proteins that share more than 50% sequence identity with RAS, including highly conserved core effector domains.Unlike RAS, the C termini of RIT and RIN lack a typical prenylation motif (CAAX, XXCC, or CXC) required for the association of RAS proteins with the plasma membrane.RIT is expressed in all tissues, whereas RIN is neuron- Activation of TRKA receptors (R-HSA-187015) 16.
Trk receptors can either be activated by neurotrophins or by two G-protein-coupled receptors (GP-CRs) although the biological relevance of GPCRs remains to be shown.
Coenzyme A (CoA) is a ubiquitous cofactor that functions as an acyl group carrier in diverse processes including fatty acid metabolism and the TCA cycle (Lipmann 1953).It is synthesized from the vitamin pantothenate in a sequence of five reactions (Daugherty et al. 2002;Leonardi et al. 2005;Robishaw and Neely 1985).These reactions all occur in the cytosol or the mitochondrial intermembrane space (Leonardi et al. 2005).A recently described transport protein appears to mediate the uptake of Coenzyme A into the mitochondrial matrix (Prohl et al. 2001).

Date
Action Author
Barker PA & Murphy RA (1992).The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins.Mol Cell Biochem, 110, 1-15.Barbacid M, Lamballe F & Klein R (1991).The trk family of oncogenes and neurotrophin receptors.Princess Takamatsu Symp, 22, 153-70.activation of phosphlipase C-gamma (PLC-gamma) and subsequent mobilization of calcium from intracellular stores are essential for neurotrophin secretion.PLC-gamma is activated through the phosphorylation by TrkA receptor kinase and this form hydrolyses PIP2 to generate inositol tris-phosphate (IP3) and diacylglycerol (DAG).IP3 promotes the release of Ca2+ from internal stores and this results in activation of enzymes such as protein kinase C and Ca2+ calmodulin-regulated protein kinases.
The H antigen is abundant in individuals with blood group O and is the essential precursor for the production of A and B antigens.A and B antigens are formed by the action of glycosyltransferases encoded by functional alleles at the ABO genetic locus.The co dominant A allele encodes A transferase, which transfers an N acetylgalactosamine (GalNAc) sugar to the H antigen forming the A antigen.Similarly, the co dominant B allele encodes B transferase, which transfers a galactose (Gal) sugar to the H antigen forming the B antigen.Individuals who have both A and B alleles form the AB antigen.Individuals who are homozygous for the recessive O allele express the H antigen but do not form A or B antigens as they lack both the glycosyltransferase enzymes for their formation.Mutant alleles of the corresponding FUT1 or FUT2 genes result in either a H-phenotype (Bombay phenotype, Oh) or a weak H phenotype (para Bombay) where the affected individual cannot form H, A or B antigens (Kaneko et al. 1997, Koda et al. 1997).The biosyntheses of the A, B and H antigens are described in this section (Ewald & Sumner 2016, Scharberg et al. 2016).References Ewald DR & Sumner SC (2016).Blood type biochemistry and human disease.Wiley Interdiscip Rev Syst Biol Med, 8, 517-535.Scharberg EA, Olsen C & Bugert P (2016).The H blood group system.Immunohematology, 32, 112-118.Shinya N, Kudo T, Okubo Y, Seno T, Iwasaki H, Kaneko M, ... Nishihara S (1997).Wide variety of point mutations in the H gene of Bombay and para-Bombay individuals that inactivate H enzyme. Blood, 90, 839-49.Johnson PH, Soejima M, Koda Y, Smart E & Kimura H (1997). Missense mutation of FUT1 and deletion of FUT2 are responsible for Indian Bombay phenotype of ABO blood group system.Biochem.Biophys.Res.Commun., 238, 21-5.
specific.They have similar signalling properties and are activated by NGF through unknown exchange factors.They signal to ERKs and p38 MAP kinase.They mainly lead to p38 activation via the BRAF-MEK kinase cascade.References Der CJ & Reuther GW (2000).The Ras branch of small GTPases: Ras family members don't fall far from the tree.Curr Opin Cell Biol, 12, 157-65.-Rich Membrane Spanning/Kidins 220) is a 220kD tetraspanning adaptor protein which becomes rapidly tyrosine phosphorylated by active Trk receptors.ARMS is another adaptor protein which is involved in the activation of Rap1 and the subsequent prolonged activation of the MAPK cascade.
p75NTR modulates axonal growth by regulating the activity of small GTPases like RHOA and RHOB, that control the state of actin polymerization.The best studied is RHOA.In its active, GTP-bound form, RHOA rigidifies the actin cytoskeleton, thereby inhibiting axonal elongation and causing growth cone collapse.Depending on the ligand that binds to it, p75NTR can either promote or inhibit axonal growth, Neurotrophin binding leads to inhibition of RHOA activity and axonal growth.Axonal growth inhibition is caused by myelin molecules named MDGIs (myelin-derived growth inhibitors), such as NOGO, MAG, OMGP.MDGIs bind to a complex made up of p75NTR and the NOGO receptor, causing RHOA activation and axonal growth inhibition.ReferencesLi HY & Zhou XF (2007).Roles of glial p75NTR in axonal regeneration.J Neurosci Res, 85, 1601-5. in electrically excitable cells such as neurons, muscles, and endocrine cells.They are initiated by transient opening of voltage dependent sodium channels, causing a rapid, large depolarization of membrane potentials that spread along the axon membrane.The action potential travels down the axon and reaches the presynaptic terminal depolarizing the membrane in the pre synaptic terminal.The depolarization causes the voltage gated Ca2+ channels to open allowing the influx of Ca2+ that signals the release of neurotransmitter into the synaptic cleft.FGFR1c ligand binding and activation(R-HSA-190373)   22.This pathway depicts the binding of an experimentally-verified range of ligands to FGFR1c.While binding affinities may vary considerably within this set, the ligands listed have been established to bring about receptor activation at their reported physiological concentrations.

submitted entities found in this pathway, mapping to 1 Reactome entities
Below is a list of the input identifiers that have been found or mapped to an equivalent element in Reactome, classified by resource.

16 of the submitted entities were found, mapping to 20 Reactome entities
These 7 identifiers were not found neither mapped to any entity in Reactome.