Discussion
Here, we have identified that undercarboxylated GRP and MGP are the
major VKD proteins causing the PXE-like phenotype and facial
dysmorphologies in VKCFD1 patients. Individualized medication with
γ-carboxylated VKD proteins to treat non-haemorrhagic phenotypes may
represent a promising strategy in future. Furthermore, our GGCX in
silico model highlights that two loss-of-function variants affect the
KH2 binding pocket in GGCX that was identified in the
present study.
The non-haemostatic phenotypes in VKCFD1 summarizes skin laxity,
skeletal and cardiac abnormalities (De Vilder, Eva Y G et al., 2017 Jan
25). However, until now it remained elusive which under-carboxylated VKD
protein causes the corresponding phenotype.
Notably, we found that GRP plays a fundamental role in the development
of the PXE-like phenotype. According to our data, the PXE-like phenotype
is caused by the biallelic deficiency to γ-carboxylate GRP. Four VKCFD1
patients were reported with a severe PXE-like phenotype, where we
detected levels of γ-carboxylated GRP between 0 - 32 % only by the
corresponding pathogenic variants. The monoallelic disability to
γ-carboxylate GRP is not leading to a skin phenotype as we see for all
heterozygous family members (parents and siblings) of patients with a
PXE-like phenotype (Table S2). These findings suggests that only the
biallelic deficiency to γ-carboxylate GRP lead to a PXE-like phenotype,
and brings us to the conclusion that the two patients being heterozygous
for either GGCX:p.(R476C) or GGCX:p.(R476H) must have an additional
defect because of the unusual early age of onset. Since the reported
genotype is also not fitting to the haemorrhagic phenotype as already
observed in a previous study (Ghosh et al., 2021), we recommend
performing next generation sequencing to potentially identify additional
variants in GGCX or other genes.
Due to the markedly reduced levels of γ-carboxylated GRP obtained in our
assay, we predict for two more patients the development of a PXE-like
phenotype later in life (Figure 2c). Unfortunately, there is currently
no treatment available because life-long administration of high doses of
K will not lead to sufficient γ-carboxylated levels of GRP as we see in
our data. However, these patients might be medicated in future by the
nanotechnology-based method of Viegas et al., where human γ-carboxylated
GRP is loaded into extracellular vesicles (Viegas et al., 2019). This
recent and smart approach was invented to inhibit vascular
calcification, which might have a high potential to treat ectopic
calcification of skin, too.
There is a low number of in total three affected patients with facial
dysmorphologies. Since loss-of-function variants in MGP lead to
Keutel syndrome, which is an autosomal recessive disorder characterized
by midfacial hypoplasia and other skeletal malformations (Munroe et al.,
1999), it was expected that GGCX variants failing to
γ-carboxylate exclusively MGP lead to midfacial hypoplasia. Tie et al.
stated that MGP is the major protein responsible for the Keutel
syndrome-like phenotype in a patient carrying
GGCX:p.(M174R+R325Q);p.(D153G) (Tie et al., 2016). We confirmed their
findings with zero levels of γ-carboxylated MGP for GGCX:p.(M174R) and
with levels of ~38% for GGCX:p.(D153G) in the other
allele. In line with these findings, low levels of γ-carboxylated MGP
below 27 % were detected for the second affected patient with the
genotype GGCX:p.(R83P);p.(R83P). However, we even observed lower levels
of γ-carboxylated MGP below 26% for three other patients that do not
have skeletal malformations (GGCX:p.(R83W);p.(Q374X),
GGCX:p.(F299S);p.(G558R), and GGCX:p.(V255M);p.(S300F)) (Table 2).
Therefore, we conclude that biallelic markedly reduced levels of
γ-carboxylated MGP are crucial but not exclusive for causing the
skeletal syndrome in VKCFD1 patients. Since the skeletal phenotype is a
congenital defect that is developed during embryogenesis, we suspect
that the maternal genotype and vitamin K uptake during pregnancy
contributes to the development of facial malformations as well. Indeed,
nutritional uptake by the mother can affect the degree of malformation,
which was shown by the study of Lanham et al., whereMgp -/- mice, whose mothers were fed on a
high-fat diet during pregnancy showed increased bone parameters as bone
length, volume, and surface compared to littermates, whose mothers were
fed on a control diet. This suggests a maternal nutrient transfer that
is influencing the outcome of the birth defects inMgp -/- mice. Moreover, Lanham et al. also
showed that GGCX expression is higher in Mgp -/-mice, whose mothers were fed on high-fat diet when compared to control
littermates (Lanham, Cagampang, & Oreffo, 2018) suggesting that GGCX
expression could be regulated through substrate availability. All
together this indicates that the nutritional uptake of vitamin K during
pregnancy might modulate the severity of those birth defects in humans
as well. Therefore, we assume that the severe hyperemesis gravidarum
with a weight loss of seven kg within the first trimester from the
mother of the patient carrying GGCX:p.(S284P);p.(W315X) had a
significant additional influence on the development of the patient´s
midfacial hypoplasia. Although this patient carries one functional
allele, where GGCX:p.(S284P) exhibit almost normal level of
γ-carboxylated MGP at high vitamin K levels, this patient was born with
midfacial hypoplasia.
Furthermore, we agree that the promoter polymorphism of VKORC1 might
modulate the severity of VKCFD1 phenotypes, especially with respect to
skeletal malformations (Watzka et al., 2014). In fact, two of the three
patients with facial hypoplasia are homozygous for the VKORC1:c.-1639 AA
genotype that is associated with 50% reduction of VKORC1 mRNA and
enzymatic activity for each allele (Rieder et al., 2005). The VKORC1
promoter genotype for the third patient is unknown. Although this
polymorphism has no impact on haemostasis within normal population, it
could influence severity of both haemorrhagic and non-haemorrhagic
phenotypes in VKCFD1 patients due to the reduced capacity to recycle
vitamin K. Therefore, we recommend to screen VKCFD1 patients also for
the VKORC1:c.-1639 genotype in future to evaluate efficiency of therapy.
Patients with the weak AA genotype might develop more severe phenotypes
and thus need higher K treatment.
In VKCFD1 patients with a cardiac phenotype, we observed for the
corresponding reported pathogenic GGCX variants that the ability
to γ-carboxylate GRP and MGP is reduced (Table S3). However, not all
patients carrying pathogenic GGCX variants causing reduced level
of γ-carboxylated GRP and MGP develop cardiac defects. Here, we assume
again that the nutritional uptake of vitamin K and the lifestyle affect
the aging phenotype as athereosclerosis, where patients with low vitamin
K uptake have a higher risk to develop a cardiac phenotype.
With respect to the structure of GGCX our model allowed to predict the
vitamin K binding site. Two pathogenic variants directly affecting this
binding site (GGCX:p.(F299S) and GGCX:p.(S300F)) resulted into
loss-of-function of all analysed VKD proteins and were also found in
another study to cause loss-of-function or diminished ability to
γ-carboxylate VKD clotting factors (Hao et al., 2020). Hao et al. showed
also that these two residues have reduced vitamin k epoxidation.
Although it is only an in silico model of GGCX and it has
limitations due to its unique structure, we assume that vitamin K
binding takes place in this region. However, future studies are required
to solve the X-ray crystal structures of GGCX for identifying the exact
vitamin K binding domain.
Even though, non-haemostatic VKCFD1 phenotypes are rare, this study
highlights the need of GGCX to sufficiently γ-carboxylate GRP and MGP
for maintaining physiological calcification. Our data will help to
further understand the diversity of known and potentially unknown VKCFD1
phenotypes. The outcome of birth defects could be potentially improved
when mothers with a GGCX defect are administered with vitamin K during
pregnancy. Personalized medication with γ-carboxylated VKD proteins as
f.e. γ-carboxylated GRP to treat non-haemostatic phenotypes may
represent a promising strategy in future.