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.