Showing papers by "Paul Coucke published in 2014"

Expressed Repeat Elements Improve RT-qPCR Normalization across a Wide Range of Zebrafish Gene Expression Studies

Abstract: The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.

Perturbation of specific pro-mineralizing signalling pathways in human and murine pseudoxanthoma elasticum

Abstract: Pseudoxanthoma elasticum (PXE) is characterized by skin (papular lesions), ocular (subretinal neovascularisation) and cardiovascular manifestations (peripheral artery disease), due to mineralization and fragmentation of elastic fibres in the extracellular matrix (ECM). Caused by mutations in the ABCC6 gene, the mechanisms underlying this disease remain unknown. The knowledge on the molecular background of soft tissue mineralization largely comes from insights in vascular calcification, with involvement of the osteoinductive Transforming Growth Factor beta (TGFβ) family (TGFβ1-3 and Bone Morphogenetic Proteins [BMP]), together with ectonucleotides (ENPP1), Wnt signalling and a variety of local and systemic calcification inhibitors. In this study, we have investigated the relevance of the signalling pathways described in vascular soft tissue mineralization in the PXE knock-out mouse model and in PXE patients. The role of the pro-osteogenic pathways BMP2-SMADs-RUNX2, TGFβ-SMAD2/3 and Wnt-MSX2, apoptosis and ER stress was evaluated using immunohistochemistry, mRNA expression profiling and immune-co-staining in dermal tissues and fibroblast cultures of PXE patients and the eyes and whiskers of the PXE knock-out mouse. Apoptosis was further evaluated by TUNEL staining and siRNA mediated gene knockdown. ALPL activity in PXE fibroblasts was studied using ALPL stains. We demonstrate the upregulation of the BMP2-SMADs-RUNX2 and TGFβ-2-SMAD2/3 pathway, co-localizing with the mineralization sites, and the involvement of MSX2-canonical Wnt signalling. Further, we show that apoptosis is also involved in PXE with activation of Caspases and BCL-2. In contrast to vascular calcification, neither the other BMPs and TGFβs nor endoplasmic reticulum stress pathways seem to be perturbed in PXE. Our study shows that we cannot simply extrapolate knowledge on cell signalling in vascular soft tissue calcification to a multisystem ectopic mineralisation disease as PXE. Contrary, we demonstrate a specific set of perturbed signalling pathways in PXE patients and the knock-out mouse model. Based on our findings and previously reported data, we propose a preliminary cell model of ECM calcification in PXE.

Type I procollagen C-propeptide defects: study of genotype-phenotype correlation and predictive role of crystal structure

Abstract: The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C-propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50-55 nucleotides upstream of the most 3' exon-exon junction lead to nonsense-mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe-to-lethal OI. Proα1(I)-C-propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)-C-propeptide is a reliable tool to predict phenotypic severity for most COL1A1-C-propeptide missense variants, whereas for COL1A2-C-propeptide variants, the phenotypic outcome is milder than predicted.

Congenital fixed dilated pupils due to ACTA2- multisystemic smooth muscle dysfunction syndrome.

Abstract: Congenital fixed dilated pupils (congenital mydriasis) is characterized by hypoplasia or aplasia of the iris muscles, with absence of iris between the collarette and pupillary border, creating a scalloped pupillary margin. This condition has been reported in a multisystemic smooth muscle cell dysfunction syndrome, combined with congenital patent ductus arteriosus, cerebrovascular disease (Moya-moya-like), coronary artery disease, thoracic aorta aneurysm, and dysfunction of smooth muscle cells in organs throughout the body. All affected individuals carry a p.R179H heterozygous mutation in the ACTA2 gene. We add to the ophthalmologic involvement with 3 more patients. Congenital fixed dilated pupils is a rare condition and should alert ophthalmologists to the possibility of the coexistence of systemic life-threatening disorders.

Arterial Tortuosity Syndrome

Abstract: Clinical characteristics Arterial tortuosity syndrome (ATS) is characterized by the following: Diagnosis/testing The diagnosis of ATS is established in a proband with generalized arterial tortuosity and biallelic (homozygous or compound heterozygous) pathogenic variants in SLC2A10. Management Treatment of manifestations: Individuals with ATS benefit from a coordinated approach of multidisciplinary specialists in a medical center familiar with ATS or similar conditions. Although hemodynamic stress on arterial walls can be reduced with use of beta-adrenergic blockers or other medications including angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin II receptor 1 (ATIIR1) antagonists such as losartan, the efficacy of these treatments has not been established in ATS and caution is warranted when using blood pressure-lowering medications in the presence of arterial stenosis (anatomic or functional due to severe tortuosity), especially renal artery stenosis. Aneurysms and focal stenoses are amenable to surgical intervention. Skeletal manifestations such as scoliosis require management by an orthopedist, and ocular manifestations require management when possible by an ophthalmologist with expertise in connective tissue disorders. Wound healing may be delayed following surgery; thus, stitches should be placed without traction and remain in place ~10 days. A supporting mesh can be used in the surgical repair of hernias to reduce recurrence risk. Surveillance: Regular cardiovascular follow up (with MRA or CT scan with 3D reconstruction from head to pelvis) starting at birth or at the time of diagnosis. Routine follow-up for: pulmonary manifestations, refractive errors and keratoconus; orthopedic complications, such as scoliosis, especially during periods of rapid growth; possible dental crowding secondary to palatal abnormalities. Agents/circumstances to avoid: Contact sports, competitive sports, and isometric exercise; scuba diving; agents that stimulate the cardiovascular system (including routine use of decongestants); tobacco use; sun tanning. Evaluation of relatives at risk: It is appropriate to evaluate the older and younger sibs of a proband with ATS in order to identify as early as possible those who would benefit from treatment and surveillance for complications. Pregnancy management: Data on the management of women with arterial tortuosity syndrome during pregnancy and delivery are limited. Preconception counseling should include possible pregnancy-associated risks to the mother (mainly aortic root dilatation and dissection) and recommendation to discontinue medications with possible teratogenic effects (e.g., angiotensin-converting enzyme inhibitors [ACE-I], angiotensin II receptor 1 antagonists [ATIIR1] such as losartan, and anticoagulant therapy) and to begin therapy with β-blockers. Genetic counseling ATS is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk relatives and prenatal testing for pregnancies at increased risk are possible if the SLC2A10 pathogenic variants in the family are known.

Absence of cardiovascular manifestations in a haploinsufficient Tgfbr1 mouse model.

Abstract: Loeys-Dietz syndrome (LDS) is an autosomal dominant arterial aneurysm disease belonging to the spectrum of transforming growth factor β (TGFβ)-associated vasculopathies. In its most typical form it is characterized by the presence of hypertelorism, bifid uvula/cleft palate and aortic aneurysm and/or arterial tortuosity. LDS is caused by heterozygous loss of function mutations in the genes encoding TGFβ receptor 1 and 2 (TGFBR1 and -2), which lead to a paradoxical increase in TGFβ signaling. To address this apparent paradox and to gain more insight into the pathophysiology of aneurysmal disease, we characterized a new Tgfbr1 mouse model carrying a p.Y378* nonsense mutation. Study of the natural history in this model showed that homozygous mutant mice die during embryonic development due to defective vascularization. Heterozygous mutant mice aged 6 and 12 months were morphologically and (immuno)histochemically indistinguishable from wild-type mice. We show that the mutant allele is degraded by nonsense mediated mRNA decay, expected to result in haploinsufficiency of the mutant allele. Since this haploinsufficiency model does not result in cardiovascular malformations, it does not allow further study of the process of aneurysm formation. In addition to providing a comprehensive method for cardiovascular phenotyping in mice, the results of this study confirm that haploinsuffciency is not the underlying genetic mechanism in human LDS.

Illumina sequencing of 15 deafness genes using fragmented amplicons

Abstract: Background Resequencing of deafness related genes using GS FLX massive parallel sequencing of PCR amplicons spanning selected genes has previously been reported as a successful strategy to discover causal variants. The amplicon lengths were designed to be smaller than the sequencing read length of GS FLX technology, but are longer than Illumina sequencing technology read lengths. Fragmentation is thus required to sequence these amplicons using high throughput Illumina technology.