[CFS-L] M.Sc. Thesis Ueland

[Dr. Marc-Alexander Fluks]

Source: University of Oslo
Date: May 18 and June 21, 2021
URL:
https://www.duo.uio.no/bitstream/handle/10852/87105/7/Ueland_MasterThesis_2021.pdf
Ref:
https://www.mn.uio.no/ibv/studier/aktuelt/mastereksamen/var-2021/marthe%20ueland.html


Genetic study of T cell receptor (TCR) in myalgic encephalomyelitis/
chronic fatigue syndrome (ME/CFS)
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Marthe Ueland
- Oslo University Hospital, Department of Medical Genetics,
Faculty of Mathematics and Natural Sciences, University of Oslo


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.

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(c) 2021 University of Oslo