Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). To date there is no effective treatment and, due to its debilitating physical and cognitive symptoms, MS is the most common cause for disability of young adults in the developed world. While the etiology of MS is yet to be elucidated, it is known to be a multifactorial disease, which develops as a consequence of genetic and environmental risk factors and their interaction. GWA studies performed to date have discovered numerous common genetic variants, which collectively do not explain the observed heritability of the disease. One plausible candidate for the explanation of missing heritability in multifactorial diseases are exceedingly rare genetic variants whose detection is now enabled by the next-generation sequencing technology (NGS). In the light of our previous research results and existing evidence linking inflammasome to MS, we investigated the mutational burden in the genes comprising the inflammasome regulatory network.
MS has been associated with increased transcription of human endogenous retroviruses (HERV) which constitute around 8 % of the human genome. Recently, several genomic loci with populationally unfixed HERV insertions were published. These represent an uncharacterized source of human genetic variability, which is interesting in the context of HERV associated diseases, such as MS. The most prominent environmental MS risk factors are infection with Epstein-Barr virus (EBV) and smoking. The aim of our research was the search for missing heritability of MS and the integration of newly found genetic risk factors with the known environmental ones.
Probands included in the research belonged to either of the three groups: Probands with sporadic MS (MSS), probands from families with multiple affected members (MSFAM), and ethnically matched control group. We employed whole exome-sequencing (WES) which is based on NGS technology as our main experimental method. The method enabled us to genotype all genetic variants in the panel of 62 genes which are known to be associated with the regulation of NLRP1/NLRP3 inflammasome. Additionally, we used bioinformatics tools to obtain the genotypes of the main risk and protective HLA alleles for MS: DRB1*15:01 and HLA-A*02:01. These two methods were applied to all 319 probands. Mutational burden analysis was weighted with populational variant frequency obtained from the “gnomAD” database and with the results of variant pathogenicity prediction algorithm: CADD. In the first phase of the research, we employed and additional method, a custom-designed targeted NGS, to systematically genotype 20 populationally unfixed HERV insertions on 102 probands. Two HERV insertions (located in the introns of RASGRF2 and PTPRN2 genes) were enriched amongst the MS probands and were genotyped on additional probands (total of 253) using the long-range PCR, which confirmed their significant association with MS. For the same set of 253 probands, we performed a quantitative estimation of EBV viremia using the real-time qPCR method. Epigenetic analysis of differential methylation of CpG loci known to be associated with smoking status was performed on 102 probands.
We have discovered a significantly increased rare variant burden in the inflammasome regulatory genes of patients with MS, which was most prominent for the exceedingly rare variants that were scored highly by the pathogenicity prediction algorithm. These variants support the overall importance of inflammasome and its regulation by interferon-β and auto/mitophagy to the etiology of MS. In the present study, we report insertion allele frequencies for 20 unfixed HERV-K insertions and demonstrate two of them to be overrepresented among the patients with MS, compared to the control cohort. The HERV-K sequence within the RASGRF2 gene was detected only in the heterozygous state and was overrepresented only in the MS patients from families with multiple affected members. The HERV-K sequence inserted within the PTPRN2 gene was overrepresented in both familial and sporadic MS patients, in both heterozygous and homozygous state, and was associated with MS according to the log-additive model applied. Both insertions were present as solo LTRs located within the intronic sequences of the respected genes. We describe an additive synergic effect between the presence of DRB1*15:01 and the absence of HLA-A*02:01 alleles in the investigated population. The analysis of EBV viremia showed a significant increase of EBV presence in the blood of MSFAM probands, however, the one amongst MSS was similar to the control group. Both MSS and the control group displayed a trend in which EBV viremia increased with probands' age at blood draw. We did not find any significant correlation between the level of EBV viremia and the investigated genomic risk factors. In the joint analysis of all measured MS risk factors, each of them contributed to the accuracy of the decision tree models.
Based on the documented interactions of the NLRP1/NLRP3 inflammasome pathway with other MS risk factors and interferon-β afferent and efferent pathways, we propose inflammasome as the central integrator of genomic and environmental risk factors for MS and put forward its importance for MS etiology.