Novel clinical and etiopathogenetic findings in Pseudoxanthoma elasticum

Soft tissue calcification in the human body can be considered part of a process of continuous  degeneration  which  we  tend  to  designate  as  “aging”.  Being  an  example  of technological wit and superb bio-engineering second to none, even the decay of this corpus can hardly be considered a random or passive event. On the contrary, calcium precipitation is regulated quite tightly by an intruiging interplay between stimulatory proteins and inhibitory factors. Thus, it has been foreseen man not to be turned into a chalk pillar in his prime years, but rather to endure a much slower process of gradual mineralization. But when this brilliant regulatory opus starts failing, the reign of human pathology is entered, confronting the body with ectopic mineralization disorders. One of the archetypes of such disease is pseudoxanthoma elasticum or PXE, in which ectopic mineralization of elastic fibres causes skin, ocular and cardiovascular complications. Despite its identification more than two centuries ago, PXE has – as many genetic disorders – always been surrounded by a haze of mystery. It is the aim of this thesis to contribute to the clinical, molecular and histopathological characterization of this fascinating disease. Through careful characterization of the PXE patient cohort followed at the Ghent Center for Medical Genetics, we were able to emphasize important clinical features, such as stroke and peripheral artery disease, as well as identifying novel phenotypical features in patients and carriers, among which were abdominal calcifications and testicular microlithiasis. Also the question of a limited or subclinical phenotype in PXE carriers was addressed and we showed them to be more prone to cardiovascular disease, next to limited ophthalmological symptoms represented by comets and comet tails. In an exploratory pilot study among over 200 consecutive ischemic stroke patients, ABCC6 hotspot analysis yielded a significant increase in ABCC6 mutations compared to a healthy reference population. This signified another example of heterozygous carriers being prone to cardiovascular and/or cerebrovascular disease and introduced the ABCC6 gene in stroke research. In single and multi-center studies, this thesis contributed to the characterization and expansion of the ABCC6 mutation spectrum, as well as the exclusion of genotype-phenotype correlations. The applied molecular strategy for mutation analysis of the ABCC6 gene proved to be an efficient and cost-effective method, yielding the highest mutation detection rate so far. Also, the continuous discussion on the mode of inheritance and in particular the existence of an autosomal dominant form of PXE could be addressed constructively. Throughout the clinical follow-up of PXE patients, we applied novel fundus imaging techniques, such as autofluorescence and infrared imaging, with substantial improvement of the diagnostic capacities of limited or subtle lesions in fundo. Through collaborative efforts, the importance of electrophysiological abnormalities – subdivided in three retinopathy phenotypes – was brought to attention. Within the span of this PhD thesis, a novel phenotype was identified and characterized both clinically and molecularly. This novel autosomal recessive disorder was coined the PXE-like syndrome, because of its resemblance with classic PXE, and was proven to be caused by mutations in the GGCX gene. Encoding the gamma-carboxylase, an enzyme important in the vitamin K (VK)-cycle, this observation implicated VK and proteins depending on this vitamin – among which are several inhibitors of mineralization – in the pathogenesis of the PXE-like syndrome and hence PXE. Through various immunohistochemical and ELISA methods, VK-dependent inhibitors of calcification were shown to be inactive or defective in these syndromes, leading to ectopic mineralization in the PXE-like syndrome but also in PXE patients. These observations could be attributed to the GGCX mutations in the PXE-like syndrome. The observation of extremely low VK serum levels – an essential co-factor for protein carboxylation in the VK-cycle – in PXE patients explained why the VK-cycle is defective in PXE. The exact link with the impaired ABCC6 transporter remains unclear, although it is tempting to think of VK or one of its associated molecules as the substrate of ABCC6. Also, these findings hold out the prospect of VK suppletion as a treatment for PXE. As such, the findings summarized in this thesis have elaborated the clinical and molecular knowledge of PXE and related disorders, and have opened novel avenues for further fundamental and applied research in the field of ectopic mineralization. Above all, they have benefitted patients and their family though a more efficient molecular diagnosis, a more to-the- point follow-up and the prospect of a treatment for their burdensome disease