Genetic study of T cell receptor (TCR) in myalgic encephalomyelitis/ chronic fatigue syndrome (ME/CFS), by Marthe Ueland in University of Oslo, June 21, 2021
Research abstract:
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling disease affecting patients physically and cognitively by e.g fatigue, post-exertional malaise (PEM), pain, memory-loss and concentration difficulties. It is currently no treatment for ME/CFS, and manifestation differs between individuals, which makes it difficult to identify its aetiology. Multiple genetic and environmental factors are believed to contribute to its development, thus categorizing it as a complex disease, which also is the case for autoimmune diseases (AID).
A hypothesis that ME/CFS is an immune-mediated disease has been suggested and are supported by various findings. Immunological alterations such as altered T cell response have been reported in patients. Additionally, has an increased occurrence of autoimmune diseases (AIDs) been observed in families with ME/CFS. The hypothesis is further supported by an identified association between human leukocyte antigen (HLA) class I and II and ME/CFS, a hallmark for most AIDs.
As HLA molecules present antigens to the T cell receptor (TCR), this receptor is of interest for further investigation. Furthermore, studies of TCRs have shown that both HLA molecules and single nucleotide polymorphisms (SNPs) located within the germline DNA can influence the gene usage in TCRs. Associations have been found between the TCR α chain
(TRA) region and immune-mediated diseases. For example has a genome-wide association study (GWAS) conducted in narcolepsy, which also has an HLA class II-association, identified associations (p<10-21) to three single nucleotide polymorphisms (SNPs) in TRA (rs1154155, rs12587781 and rs1263646), which was the first documented involvement of this region in disease. An additional small GWAS in ME/CFS showed association between three SNPs in TRA (rs17255510, rs11157573 and rs10144138) and the disease (adjusted p<0.05).
The aim of this thesis was to find methods that can be used to study genetic variants in the T cell receptor α region (TRA) to identify possible associations with ME/CFS. This was done by genotyping and sequencing. Association analysis of 30 SNPs genotyped using Illumina Immunochip (Ichip) and Taqman assays in a Norwegian cohort of 408 ME/CFS cases and 721 controls failed to show any association between TRA and ME/CFS. Since these included two of the SNPs previously associated with ME/CFS (rs17255510 and rs11157573), we did not replicate the findings.
Analysis of Ichip´s coverage of the TRA gene showed that it was inadequate with only 27 SNPs covered in this region, although 737 has been identified in the 1000 genomes CEU dataset. The TCR genetic regions are generally understudied due to homology and repetitive regions, which is problematic to cover with existing methods. Hence, two sequencing protocols were established in the TRA region. PacBio´s No-amp targeted sequencing utilizing the CRISPR-Cas9 system with SMRT sequencing was tested for fragments ranging from 4.8 to 20.1 kb, however, the highest read depth was obtained for fragments <6 kb. We conclude that this protocol is not suited for screening but can be a good complement to other sequencing methods. Long-range PCR with Illumina Miseq sequencing resulted in read depth able to detect genetic variant for some fragments, however, the approach required a lot of optimization. The obtained sequences were not studied in detail during this work and would therefore be of interest to investigate further to identify genetic variants. Future studies in this region would include targeted enrichment using capture probes.
In conclusion, we did not detect any association between ME/CFS and TRA, however, we revealed that the genetic variants tested thus far does not capture the genetic variation in this region. Furthermore, the sequencing protocols tested pave the way for further optimization and characterization of TRA by sequencing.
