The Abcc6a Knockout Zebrafish Model as a Novel Tool for Drug Screening for Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by pathogenic variants in the ABCC6 gene. Though complications of the disease can be treated, PXE itself remains currently intractable. A strategy for rapid and cost-effective discovery of therapeutic drugs would be to perform chemical compound screening using zebrafish, but this approach remains to be validated for PXE. In this paper, we validate a stable CRISPR/Cas9 abcc6a knockout zebrafish model-which has spinal column hypermineralization as its primary phenotypic feature-as a model system for compound screening in ectopic mineralization. We evaluated the anti-mineralization potential of five compounds, which had (anecdotal) positive effects reported in Abcc6 knockout mice and/or PXE patients. Abcc6a knockout zebrafish larvae were treated from 3 to 10 days post-fertilization with vitamin K1, sodium thiosulfate, etidronate, alendronate or magnesium citrate and compared to matching controls. Following alizarin red S staining, alterations in notochord sheath mineralization were semiquantified and found to largely congrue with the originally reported outcomes. Our results demonstrate that the use of this abcc6a knockout zebrafish model is a validated and promising strategy for drug discovery against ectopic mineralization.

Severe early-onset manifestations of pseudoxanthoma elasticum resulting from the cumulative effects of several deleterious mutations in ENPP1, ABCC6 and HBB: transient improvement in ectopic calcification with sodium thiosulfate.

Pseudoxanthoma elasticum (PXE) is a rare disorder characterized by fragmentation and progressive calcification of elastic fibres in connective tissues. Overlap has been reported between the inherited PXE phenotype associated with ENPP1, ABCC6 or NT5E mutations and acquired PXE clinical manifestations associated with haemoglobinopathies induced by HBB mutations. No treatment is currently available for PXE. A young boy presented with severe early-onset systemic calcifications occurring in the skin as elastosis perforans serpiginosa (EPS) and in the arteries, causing mesenteric and limb ischaemia. Analyses revealed deleterious ABCC6, ENPP1 and HBB mutations. The diagnosis of severe PXE was retained and we have coined the term 'PXE+ syndrome' to describe the cumulative effects of the various mutations in this uncommon phenotype. Given the severity, rapid progression and a potentially fatal prognosis, intravenous sodium thiosulfate (STS) was initiated at 25 g three times weekly for 6 months. Numerous side-effects prompted dosage adjustment to 10 g intravenously daily. Treatment efficacy was evaluated at 6 months. Asthaenia, anorexia and pre-/postprandial pain had subsided, entailing weight gain. Abdominal EPS had diminished. Calcific stenosis of the coeliac and mesenteric arteries was no longer detectable on arterial ultrasonography. Follow-up revealed only transient efficacy of STS. Discontinuation of treatment to evaluate the persistence of effects resulted in relapse of the initial symptomatology after 4 months. STS efficacy is conceivably due to strong antioxidant properties and chelation of calcium to form soluble calcium thiosulfate complexes. This case is suggestive of PXE+ syndrome for which STS may represent potential treatment in severe cases. What's already known about this topic? Generalized arterial calcification of infancy may occur in association with ABCC6 mutations and pseudoxanthoma elasticum (PXE) can be linked to ENPP1 mutations. A PXE-like phenotype has also been reported in a subset of patients with inherited haemoglobinopathies, namely sickle cell disease or ß-thalassaemia, related to HBB mutations. To date, there is still no cure for PXE. What does this study add? We report a severe case of PXE resulting from the cumulative effects of several deleterious mutations in ENPP1, ABCC6 and HBB. We suggest the term 'PXE+ syndrome' to describe such patients. Sodium thiosulfate therapy could represent a potential option in severe cases of PXE+ syndrome.

Cellular signaling in pseudoxanthoma elasticum: an update.

Pseudoxanthoma elasticum is an autosomal recessive genodermatosis with variable expression, due to mutations in the ABCC6 or ENPP1 gene. It is characterized by elastic fiber mineralization and fragmentation, resulting in skin, eye and cardiovascular symptoms. Significant advances have been made in the last 20 years with respect to the phenotypic characterization and pathophysiological mechanisms leading to elastic fiber mineralization. Nonetheless, the substrates of the ABCC6 transporter - the main cause of PXE - remain currently unknown. Though the precise mechanisms linking the ABCC6 transporter to mineralization of the extracellular matrix are unclear, several studies have looked into the cellular consequences of ABCC6 deficiency in PXE patients and/or animal models. In this paper, we compile the evidence on cellular signaling in PXE, which seems to revolve mainly around TGF-ßs, BMPs and inorganic pyrophosphate signaling cascades. Where conflicting results or fragmented data are present, we address these with novel signaling data. This way, we aim to better understand the up- and down-stream signaling of TGF-ßs and BMPs in PXE and we demonstrate that ANKH deficiency can be an additional mechanism contributing to decreased serum PPi levels in PXE patients.

Generation and Validation of a Complete Knockout Model of abcc6a in Zebrafish.

Pseudoxanthoma elasticum is an ectopic mineralization disease due to biallelic ABCC6 mutations. As no treatment options are currently available, a reliable zebrafish model is invaluable for high throughput compound screening. However, data from previously reported knockdown and mutant zebrafish models for abcc6a, the functional orthologue of ABCC6, showed phenotypic discrepancies. To address this, we developed a complete abcc6a knockout model using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 and compared its phenotype to that of a mutant model (Sa963) and a splice junction morpholino model. Our data showed that abcc6a is not required for embryonic survival, but rather that it has an essential role in controlling mineralization. The three models developed very similar hypermineralization of spine and ribs starting embryonically and progressing in adulthood with development of scoliosis. Our results indicate a direct relation between loss of abcc6a expression and dysregulated osteogenesis. As such, our models recapitulate part of the human phenotype in which ectopic mineralization and pro-osteogenic signaling have been reported. Because of its reproducibility in three models and its ease of quantification, we consider this phenotype to be unequivocally the result of abcc6 deficiency and, as such, an excellent readout for drug screening purposes and multiplex mutagene analysis.

Pharmacogenomics in children: advantages and challenges of next generation sequencing applications.

Pharmacogenetics is considered as a prime example of how personalized medicine nowadays can be put into practice. However, genotyping to guide pharmacological treatment is relatively uncommon in the routine clinical practice. Several reasons can be found why the application of pharmacogenetics is less than initially anticipated, which include the contradictory results obtained for certain variants and the lack of guidelines for clinical implementation. However, more reproducible results are being generated, and efforts have been made to establish working groups focussing on evidence-based clinical guidelines. For another pharmacogenetic hurdle, the speed by which a pharmacogenetic profile for a certain drug can be obtained in an individual patient, there has been a revolution in molecular genetics through the introduction of next generation sequencing (NGS), making it possible to sequence a large number of genes up to the complete genome in a single reaction. Besides the enthusiasm due to the tremendous increase of our sequencing capacities, several considerations need to be made regarding quality and interpretation of the sequence data as well as ethical aspects of this technology. This paper will focus on the different NGS applications that may be useful for pharmacogenomics in children and the challenges that they bring on.

Visceral and testicular calcifications as part of the phenotype in pseudoxanthoma elasticum: ultrasound findings in Belgian patients and healthy carriers.

Occasionally calcifications in abdominal organs, breasts and testicles have been reported in pseudoxanthoma elasticum (PXE) patients. In the present study, an ultrasound evaluation was performed of the abdomen and--in male patients--of the testicles in 17 PXE patients and 17 heterozygous carriers. Blood samples were taken to evaluate calcium load, liver and kidney function. Calcifications in liver, kidneys and spleen were detected in 59% of the patients and in 23.5% of healthy carriers. Parameters of kidney and liver function were normal in both groups, suggesting that the calcifications have no direct effect on organ function. Testicular ultrasound revealed parenchymous calcifications in all males investigated. Widespread, small hyperechogenic foci resembling testicular microlithiasis were seen. In some carriers, focal calcifications were identified. The current data suggest that visceral and testicular calcifications are part of the phenotype of PXE patients. Their presence in some of the healthy carriers are suggestive of subclinical manifestations in these relatives. The natural history and long-term effects of the parenchymal calcifications remain to be elucidated. As testicular microlithiasis may be associated with a higher risk for malignancy, regular clinical and ultrasound follow-up seems indicated in these patients.