DISCUSSION
In our patient, seizure onset was in early infancy, yet she displayed
neurological and behavioural abnormalities since birth and hence the
label DEE. Severely impaired cognitive and motor development was already
notable within the first year of life and acquired microcephaly
reflected atrophy and/or cerebral underdevelopment in the context of an
epileptic and developmental disorder.
We here provided evidence that dysfunction of CHRM1 might cause DEE, or
CHRM1 encephalopathy. While mutations in the various genes encoding
ionotropic nicotinic acetylcholine receptor subunits CHRNA4 ,CHRNB2 and CHRNA2 are typically associated with autosomal
dominant nocturnal frontal lobe epilepsy (Steinlein et al., 1995; De
Fusco et al., 2000; Aridon et al., 2006), to our knowledge this is the
first instance where the metabotropic muscarinic receptors are linked to
an epileptic disorder. Admittedly, this is based only in the fact that
we have identified one de novo mutation in a single patient.
However, we believe that this mutation is pathogenic since it affects a
conserved residue in all CHRM proteins that is present in an important
structural element of the transmembrane segment 6 (TM6). The TM6 suffers
a small rotation and an outward displacement during receptor activation
that allows the G protein to engage the receptor core (Maeda et al.,
2019). Thus, it could be that the p.P380L mutation impairs receptor
activation as our data suggest. In addition, expression of this mutant
protein in transfected cells at the membrane is reduced possibly due to
a folding defect. We reasoned that, based on the fact that the mutant
protein is expressed at low levels, it is very difficult to consider
that the mutant protein may exert a dominant-negative effect. Rather we
support the hypothesis that the patient may suffer a reduction of
cholinergic activity due to haploinsufficiency. This hypothesis is in
agreement with the fact that a minor reduction of KCNQ activity, a known
target of muscarinic regulation, is enough to cause an epileptic
phenotype (Jentsch, 2000).
How a reduction in cholinergic activity in humans may lead to an early
epileptic phenotype? One of the best known targets of muscarinic
regulation is the M current formed by KCNQ channels through
Gq/11 mediated protein signals that increase
phospholipase C-beta activity, which result in consumption of
phosphatidylinositol 4,5-bisphosphate (PIP2) resulting in KCNQ
inhibition. In this pathway, loss of function mutations in KCNQ channels
or PLCB causes epileptic encephalopathy. Thus, considering this
well-known pathway, one simple hypothesis is that muscarinic inhibition
might result in increased KCNQ activity, which may affect neuronal
excitability properties. However, gain-of-function variants in KCNQ2 do
not show epileptic seizures (Miceli et al., 2015). Thus, modulation of M
current by CHRM1-PLCB1 might not be the only reason of pathogenesis.
CHRM1 might regulate many different targets including ion channels
depending on the developmental stage and in cell-specific manner. For
example, in the hippocampus, activation of CHRM1 might stimulate M
current, G-protein coupled inwardly rectifying potassium channels and
TRPC channels in dentate-gyrus granule cells while it may inhibit M
current in CA1 neurons (Carver and Shapiro, 2019). On the other hand,
activation of CHRM1 might inhibit calcium-dependent small conductance
potassium (SK) channels leading to NMDA receptor disinhibition (Tigaret
et al., 2018).
It is noteworthy that, although CHRM1 is nearly not expressed in the
cerebellum (Bakker et al., 2015), our patient developed a prominent
cerebellar atrophy suggesting that a defective CHRM1-mediated
cholinergic activity may have resulted particularly damaging for
Purkinje cells.
In summary, our work further suggests that muscarinic activity in the
brain might affect multiple processes regulating seizure susceptibility
and neuronal development and therefore, CHRM1 can be proposed as
a novel gene associated to DEE phenotype.