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

Publisher Copyright: © 2023, The Author(s).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.Peer reviewe

Purification of H₆-tagged MHC class II complexes.

<p>A) Preparative purification of DRB1*04∶01 refolded with Hep C-NS3_{218–235(H6)} (YAAQGYKVLVLNPSVAATHHHHHH) on IMAC column, a shallow gradient of 20% 8 M Urea, 25 mM Tris, pH8, and 1 M NaCl (buffer B) elutes peak 1 whereas peak 2 elutes by raising the concentration of buffer B to 100%. B) SDS-PAGE analysis of eluted fractions, the αchain travels as the upper band. C) Functional LOCI assay on load, run through, and peak fractions. Notice the absence of peptide-loaded MHC class II molecules in the run through (RT) and in peak 1. D) Assessing the level of biotinylation of a DRB1*11∶01 chain by incubation with increasing amounts of avidin followed by SDS-PAGE analysis.</p

MHC class II tetramer stainings of Ag-specific CD4+ T cells.

<p>PBMCs from four donors were <i>in vitro</i> cultured with a mix of three different peptides (HA_307–318, and IE1_211–225, and MP_60–73) and analyzed with ten different tetramers. The tetramer used is indicated horizontally above each column of flow cytometry plots. Each row of these plots corresponds to one donor, where the HLA-DR-profile and cytokine response of this donor against the particular peptide is indicated. Plots representing tetramer labeling where the peptide/HLA class II components are deemed relevant (i.e. donor responded to the peptide and possessed the HLA class II molecule) are framed in bold. The frequencies (%) of CD4+ T cells that stain with the particular tetramers are indicated in each plot. A) A total of three different tetramers were made with HA_{307–318(H6)} and analyzed for the four donors. B) Three different tetramers were made with IE1_{211–225(H6)} and analyzed for the four donors. C) A total of four tetramers were made with MP_60–73(H6) and analyzed for the four donors.</p

Peptide-HLA class II affinity measurements (in nM) of H₆- vs. non-H₆-tagged peptides with four different HLA class II molecules.

<p>NB, non-binding peptide.</p

Head to head comparison of MHC class II tetramers generated with H₆-tagged peptides versus non-H₆-tagged peptides.

<p>PBMCs <i>in vitro</i> cultured with a yellow fever epitope were stained with two tetramers carrying the same peptide-HLA class II specificity (Yellow fever virus CapsidC_49–63 restricted to HLA-DRB1*01∶01). Monomeric peptide-HLA class II complexes generated with H₆-tagged peptide were tetramerized with PE-conjugated SA (y-axis). Monomeric peptide-HLA class II complexes generated with non-H₆-tagged peptide were tetramerized with APC-conjugated SA (x-axis). All CD4+ T cells stained with non-H₆-tagged tetramer are also stained with the H₆-tagged tetramer.</p