Long read sequencing characterises a novel structural variant, revealing underactive AKR1C1 with overactive AKR1C2 .. severe fatigue, 2023, Oakley et

Hoopoe

Hoopoe

Senior Member (Voting Rights)
Long read sequencing characterises a novel structural variant, revealing underactive AKR1C1 with overactive AKR1C2 as a possible cause of unexplained severe fatigue


Abstract
Background
Causative genetic variants cannot yet be found for many disorders with a clear heritable component, including chronic fatigue disorders like myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These conditions may involve genes in dicult-to-align genomic regions that are refractory to short read approaches. Structural variants in these regions can be particularly hard to detect or dene with short reads, yet may account for a signicant number of cases. Long read sequencing can overcome these difficulties but so far little data is available regarding the specic analytical challenges inherent in such regions, which need to be taken into account to ensure that variants are correctly identied. Research into chronic fatigue disorders faces the additional challenge that the heterogeneous patient population likely encompasses multiple aetiologies with overlapping symptoms, rather than a single disease entity, such that each individual abnormality may lack statistical signicance within a larger sample. Better delineation of patient subgroups is needed to target research and treatment.

Methods
We use nanopore sequencing in a case of unexplained severe fatigue to identify and fully characterise a large inversion in a highly homologous region spanning the AKR1C gene locus, which was indicated but could not be resolved by short-read sequencing. We then use GC-MS/MS serum steroid analysis to investigate the functional consequences.

Results
Several commonly used bioinformatics tools are confounded by the homology but a combined approach including visual inspection allows the variant to be accurately resolved. The DNA inversion appears to increase the expression of AKR1C2 while limiting AKR1C1 activity, resulting in a relative increase of inhibitory neurosteroids and impaired progesterone metabolism.

Conclusions
This study provides an example of how long read sequencing can improve diagnostic yield in research and clinical care, and highlights some of the analytical challenges presented by regions containing tandem arrays of genes. It also proposes a novel gene associated with a specic disease aetiology that may be an underlying cause of complex chronic fatigue and possibly other conditions too. It reveals biomarkers that could be assessed in a larger cohort, potentially identifying a subset of patients who might respond to treatments suggested by the aetiology.

Link to free full text https://assets.researchsquare.com/f...-e3fe-41f8-a5f0-88fa9c1b5543.pdf?c=1695308213

The discussion and results are interesting.
 
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For me the take home message is

a) this seems to be more evidence that hormone dysfunction is a possible path to ME/CFS.
b) structural genomic variants are hard to detect with the usual methods, and can sometimes explain an illness.
c) this is also more evidence that in-depth genetic testing of people with ME/CFS and a family history of fatigue and/or ME/CFS could be a useful research project. The abnormalities are there, we just need to find them.
 
Hoopoe said:
Causative genetic variants cannot yet be found for many disorders with a clear heritable component, including chronic fatigue disorders like myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).
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Perhaps I'm misreading this, or it can be chalked up to awkward wording, but I am unaware that anyone has irrefutably demonstrated yet that ME/CFS has a clear heritable component.
 
DNA was extracted from 1 mL of whole blood with the Nanobind CBB Big DNA kit (Circulomics NB-900-001-01). After extraction, the genomic DNA (gDNA) was homogenised by 3-10 passes of needle shearing (26G) and 1h of incubation at 50°C. The gDNA was then split into two aliquots. 4 μg of gDNA were fragmented to ~50kb with the Megaruptor 3 (Diagenode, E07010001 and E07010003) and 10 μg were fragmented to 20-25kb with a gTUBE (Covaris, 520079). All samples were depleted of short DNA fragments (less than 10kb long) with the SRE kit (Circulomics, SS-100-101-01). DNA size distributions were assessed at each step on a FemtoPulse system (Agilent, M5330AA and FP-1002-0275) and all samples were quantified on a Qubit fluorometer (Invitrogen, Q33226).
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I have no idea what any of those devices are. We're living in the future.

They make no mention of PEM, but that her diagnosis of ME/CFS was eventually removed. Maybe she didn't have PEM? It's an interesting paper nonetheless.
 
duncan said:
Perhaps I'm misreading this, or it can be chalked up to awkward wording, but I am unaware that anyone has irrefutably demonstrated yet that ME/CFS has a clear heritable component.
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The text (as opposed to the abstract) is more nuanced and accurate:
Disorders of unexplained chronic fatigue (CF) such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) show strong evidence of heritability
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There was previously a diagnosis of ME/CFS for some years but that label can be a barrier to getting appropriate treatment so was removed after the later diagnosis of other conditions known to cause persistent fatigue.
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Of particular note was that certain steroid medications provoked an atypical reaction resembling the effects of benzodiazepines, leading to a hypothesis of excessive neurosteroid-mediated activation of the GABAAR. The patient reported that a similar but less acute sense of sedation, distinct from the fatigue, had started some years previously. It had originally occurred only in the latter stages of the menstrual cycle but had progressively spread throughout the cycle until it is now constantly present, although still with some cyclic variation.
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Note, despite this seeming to be a genetic problem, the symptoms started after an infection at age 15. The mother may also have the genetic variation but only had debilitating fatigue for a period of time.
The family history included unexplained fatigue in several family members, albeit to a much lesser degree, including a period of quite debilitating fatigue in the mother’s early 20s and a maternal second cousin diagnosed with ME/CFS.
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Another important message from this paper is that we may have been missing highly significant genetic abnormalities in ME/CFS because of technical limitations.

Here, we demonstrate the power of LRS to fully characterise a large inversion in a difficult-to-align region that is all but invisible to short reads.
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Many families have an at least partially inherited form of ME/CFS but despite this we know very little about the genetic basis of the illness. Maybe the genetic causes in these cases are in regions of the genome that are difficult to work with.
 
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ME/CFS Skeptic said:
Looks like it was based on data from only 1 patient?
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Just wondering if looking for rare cases like this* could be a way to identify the disease mechanism for large(r) groups of people with ME/CFS?
*"increase the expression of AKR1C2 while limiting AKR1C1 activity, resulting in a relative increase of inhibitory neurosteroids and impaired progesterone metabolism."
 
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