Identification of novel risk loci and causal insights for sporadic Creutzfeldt-Jakob disease: a genome-wide association study

Background: Human prion diseases are rare and usually rapidly fatal neurodegenerative disorders, the most common being sporadic Creutzfeldt-Jakob disease (sCJD). Variants in the PRNP gene that encodes prion protein are strong risk factors for sCJD but, although the condition has similar heritability to other neurodegenerative disorders, no other genetic risk loci have been confirmed. We aimed to discover new genetic risk factors for sCJD, and their causal mechanisms. Methods: We did a genome-wide association study of sCJD in European ancestry populations (patients diagnosed with probable or definite sCJD identified at national CJD referral centres) with a two-stage study design using genotyping arrays and exome sequencing. Conditional, transcriptional, and histological analyses of implicated genes and proteins in brain tissues, and tests of the effects of risk variants on clinical phenotypes, were done using deep longitudinal clinical cohort data. Control data from healthy individuals were obtained from publicly available datasets matched for country. Findings: Samples from 5208 cases were obtained between 1990 and 2014. We found 41 genome-wide significant single nucleotide polymorphisms (SNPs) and independently replicated findings at three loci associated with sCJD risk; within PRNP (rs1799990; additive model odds ratio [OR] 1·23 [95% CI 1·17-1·30], p=2·68 × 10-15; heterozygous model p=1·01 × 10-135), STX6 (rs3747957; OR 1·16 [1·10-1·22], p=9·74 × 10-9), and GAL3ST1 (rs2267161; OR 1·18 [1·12-1·25], p=8·60 × 10-10). Follow-up analyses showed that associations at PRNP and GAL3ST1 are likely to be caused by common variants that alter the protein sequence, whereas risk variants in STX6 are associated with increased expression of the major transcripts in disease-relevant brain regions. Interpretation: We present, to our knowledge, the first evidence of statistically robust genetic associations in sporadic human prion disease that implicate intracellular trafficking and sphingolipid metabolism as molecular causal mechanisms. Risk SNPs in STX6 are shared with progressive supranuclear palsy, a neurodegenerative disease associated with misfolding of protein tau, indicating that sCJD might share the same causal mechanisms as prion-like disorders. Funding: Medical Research Council and the UK National Institute of Health Research in part through the Biomedical Research Centre at University College London Hospitals National Health Service Foundation Trust

Comparison of differential methylation in parental strains and allele-specific methylation in F1 crosses.

<p>(A) Venn diagram showing the number of CpGs tested for differential methylation in the parental strains ONLY or for allele-specific methylation ONLY (grey areas) and the fraction of CpGs tested for BOTH (intersection, blue). The light blue area shows the number of CpGs significantly differentially methylated between the parentals AND/OR showing allele-specific methylation. Areas are not proportional. CpGs on the X chromosome and the mitochondrial chromosome have been excluded. (B) Overlap of differential methylation between the parental strains and allele specific methylation in the F1s. In total, 52,410 out of the 1,705,718 CpG dinucleotides analysed in both the parentals and the F1s (intersection, blue in (A)) showed significant differential methylation. The Venn diagram shows the fraction of CpGs that were differentially methylated between the parents ONLY (purple), between the alleles in the F1s ONLY (orange) or in BOTH (green). The theoretical distribution of frequencies assuming random overlap between the parental and the F1 data sets are shown in italics (<i>X²</i> = 135276.2, <i>P</i><4.9×10⁻³²⁴). The overlap of the two sets is >15 fold higher than expected by chance. (C) Frequency of CpGs with a SNP within 50 base pairs for CpGs showing differential methylation between the parentals only (purple), the F1 alleles only (orange) and both (green) compared to all CpGs analysed in the parentals and the F1s (blue) and the whole genome (red).</p

Hierarchical clustering and principal component analysis of CpG methylation profiles obtained in the F1 reciprocal crosses.

<p>Dendrograms showing the results of clustering the CpG methylation profiles obtained in each of the F1 animals before (A) and after (C) phasing the read data by parental genotype. The prefix of the phased F1 profiles in (C) denotes the reciprocal cross (bnxshr, shrxbn) and the suffix the genotype of the phased read set (bn = Brown Norway, shr = Spontaneously Hypertensive Rat). Numbers after the reciprocal cross prefix denote biological replicates. Profiles were clustered by the Ward's method using the pairwise euclidean distance between the profiles as the distance metric. Panels B and D show the projection of methylation profiles onto the 1^st principal component (PC). Replicates for each cross-genotype combination are separated along the y-axis. The prefix of the label denotes the reciprocal cross (bnxshr, shrxbn), the suffix denotes the parental genotype (bn, shr). While the 1^st PC does not separate crosses in the unphased data it provides complete separation by parental genotype in the phased data. Only CpGs with at least 5× coverage in each replicate/phased read set were included in the analysis and CpG positions affected by SNPs/indels were removed prior to clustering and principal component analysis.</p

Summary of differential methylation by source of variation.

<p>*Strand specific methylation.</p><p>Summary of differential methylation by source of variation.</p

Meth-QTL amplicon linkage and methylation.

a<p>Gene ID indicates the closest gene to the amplicon.</p>b<p>(CGI) within or overlaps with a CpG island.</p>c<p>(WGBS) whole genome bisulfite sequencing, methylation percentage in BN and SHR strains, or difference between the strains (BN-SHR).</p>d<p>(NSL) No significant linkage is defined as P>0.05 after a minimum of 1000 permutations of the linkage data.</p><p>Assay IDs are provided where there are two identical gene IDs.</p><p>Meth-QTL amplicon linkage and methylation.</p