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1.  Gene panel sequencing in heritable thoracic aortic disorders and related entities – results of comprehensive testing in a cohort of 264 patients 
Heritable Thoracic Aortic Disorders (H-TAD) may present clinically as part of a syndromic entity or as an isolated (nonsyndromic) manifestation. About one dozen genes are now available for clinical molecular testing. Targeted single gene testing is hampered by significant clinical overlap between syndromic H-TAD entities and the absence of discriminating features in isolated cases. Therefore panel testing of multiple genes has now emerged as the preferred approach. So far, no data on mutation detection rate with this technique have been reported.
We performed Next Generation Sequencing (NGS) based screening of the seven currently most prevalent H-TAD-associated genes (FBN1, TGFBR1/2, TGFB2, SMAD3, ACTA2 and COL3A1) on 264 samples from unrelated probands referred for H-TAD and related entities. Patients fulfilling the criteria for Marfan syndrome (MFS) were only included if targeted FBN1 sequencing and MLPA analysis were negative.
A mutation was identified in 34 patients (13%): 12 FBN1, one TGFBR1, two TGFBR2, three TGFB2, nine SMAD3, four ACTA2 and three COL3A1 mutations. We found mutations in FBN1 (N = 3), TGFBR2 (N = 1) and COL3A1 (N = 2) in patients without characteristic clinical features of syndromal H-TAD. Six TAD patients harboring a mutation in SMAD3 and one TAD patient with a TGFB2 mutation fulfilled the diagnostic criteria for MFS.
NGS based H-TAD panel testing efficiently reveals a mutation in 13% of patients. Our observations emphasize the clinical overlap between patients harboring mutations in syndromic and nonsyndromic H-TAD related genes as well as within syndromic H-TAD entities, justifying a widespread application of this technique.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-014-0221-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4326194  PMID: 25644172
Heritable Thoracic Aortic Disorders – next generation sequencing – Aneurysm; Dissecting/genetics – mutation detection rate
2.  Expressed Repeat Elements Improve RT-qPCR Normalization across a Wide Range of Zebrafish Gene Expression Studies 
PLoS ONE  2014;9(10):e109091.
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.
PMCID: PMC4195698  PMID: 25310091
3.  Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans 
Human mutation  2007;28(3):209-221.
Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype–phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events.
PMCID: PMC4144349  PMID: 17078022
osteogenesis imperfecta; type I collagen; genotype–phenotype; proteoglycan binding; COL1A1; COL1A2
4.  Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter 
PLoS ONE  2014;9(7):e102779.
The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein (ABCC6), primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS) and leukotriene C4 (LTC4) were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3) was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.
PMCID: PMC4111409  PMID: 25062064
Human mutation  2012;34(1):111-121.
Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5 and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in 2 probands, whereas 9 probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastro-intestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor beta (TGFβ) signaling. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs*27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFβ signaling. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.
PMCID: PMC4105850  PMID: 22829427
LTBP4; FBLN5; Urban-Rifkin-Davis syndrome; fibrillin; cutis laxa; recessive
6.  Perturbation of specific pro-mineralizing signalling pathways in human and murine pseudoxanthoma elasticum 
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.
PMCID: PMC4022264  PMID: 24775865
Pseudoxanthoma elasticum; Ectopic mineralization; Elastic fibres; Osteogenic signalling pathway; BMP2-SMADs-RUNX2; TGFβ signalling; Canonical Wnt pathway; Apoptosis; Endoplasmic reticulum stress
7.  Absence of Cardiovascular Manifestations in a Haploinsufficient Tgfbr1 Mouse Model 
PLoS ONE  2014;9(2):e89749.
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.
PMCID: PMC3933654  PMID: 24587008
8.  Novel MYH11 and ACTA2 mutations reveal a role for enhanced TGFβ signaling in FTAAD 
International journal of cardiology  2011;165(2):314-321.
Thoracic aortic aneurysm / dissection (TAAD) is a common phenotype that may occur as an isolated manifestation or within the constellation of a defined syndrome. In contrast to syndromic TAAD, the elucidation of the genetic basis of isolated TAAD has only recently started. To date, defects have been found in genes encoding extracellular matrix proteins (fibrillin-1, FBN1; collagen type III alpha 1, COL3A1), proteins involved in transforming growth factor beta (TGFβ) signaling (TGFβ receptor 1 and 2, TGFBR1/2; and SMAD3) or proteins that build up the contractile apparatus of aortic smooth muscle cells (myosin heavy chain 11, MYH11; smooth muscle actin alpha 2, ACTA2; and MYLK).
Methods and results
In 110 non-syndromic TAAD patients that previously tested negative for FBN1 or TGFBR1/2 mutations, we identified 7 ACTA2 mutations in a cohort of 43 familial TAAD patients, including 2 premature truncating mutations. Sequencing of MYH11 revealed an in frame splice-site alteration in one out of two probands with TAA(D) associated with PDA but none in the series of 22 probands from the cohort of 110 patients with non-syndromic TAAD. Interestingly, immunohistochemical staining of aortic biopsies of a patient and a family member with MYH11 and patients with ACTA2 missense mutations showed upregulation of the TGFβ signaling pathway.
MYH11 mutations are rare and typically identified in patients with TAAD associated with PDA. ACTA2 mutations were identified in 16% of a cohort presenting familial TAAD. Different molecular defects in TAAD may account for a different pathogenic mechanism of enhanced TGFβ signaling.
PMCID: PMC3253210  PMID: 21937134
thoracic aortic aneurysm; myosin heavy chain 11; smooth muscle α-actin; TGFβ signaling
9.  Ehlers-Danlos Syndrome Type VIII: A Rare Cause of Leg Ulcers in Young Patients 
Ehlers-Danlos syndrome type VIII (EDS-VIII) is a very rare autosomal dominant disease characterized by early-onset periodontitis associated with features of Ehlers-Danlos syndrome. We report a 32-year-old man whose chronic leg ulcer led to the diagnosis of EDS-VIII. He had severe periodontitis with complete loss of permanent teeth and skin fragility with thin skin, atrophic scars, and brownish atrophic pretibial plaques. Leg ulcer is not a prominent feature of EDS-VIII. We suggest adding EDS-VIII to the list of rare diseases accounting for chronic leg ulcers, if this case report prompts others to report leg ulcers associated with EDS-VIII.
PMCID: PMC3809367  PMID: 24198978
10.  Deficiency for the ER-stress transducer OASIS causes severe recessive osteogenesis imperfecta in humans 
Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous brittle bone disorder. Whereas dominant OI is mostly due to heterozygous mutations in either COL1A1 or COL1A2, encoding type I procollagen, recessive OI is caused by biallelic mutations in genes encoding proteins involved in type I procollagen processing or chaperoning. Hitherto, some OI cases remain molecularly unexplained. We detected a homozygous genomic deletion of CREB3L1 in a family with severe OI. CREB3L1 encodes OASIS, an endoplasmic reticulum-stress transducer that regulates type I procollagen expression during murine bone formation. This is the first report linking CREB3L1 to human recessive OI, thereby expanding the OI gene spectrum.
PMCID: PMC3850743  PMID: 24079343
Osteogenesis imperfecta; Type I collagen; OASIS; CREB3L1; Endoplasmic reticulum stress
11.  Helical mutations in type I collagen that affect the processing of the amino-propeptide result in an Osteogenesis Imperfecta/Ehlers-Danlos Syndrome overlap syndrome 
Whereas mutations affecting the helical domain of type I procollagen classically cause Osteogenesis Imperfecta (OI), helical mutations near the amino (N)-proteinase cleavage site have been suggested to result in a mixed OI/Ehlers-Danlos syndrome (EDS)-phenotype.
We performed biochemical and molecular analysis of type I (pro-) collagen in a cohort of seven patients referred with a clinical diagnosis of EDS and showing only subtle signs of OI. Transmission electron microscopy of the dermis was available for one patient.
All of these patients harboured a COL1A1 / COL1A2 mutation residing within the most N-terminal part of the type I collagen helix. These mutations affect the rate of type I collagen N-propeptide cleavage and disturb normal collagen fibrillogenesis. Importantly, patients with this type of mutation do not show a typical OI phenotype but mainly present as EDS patients displaying severe joint hyperlaxity, soft and hyperextensible skin, abnormal wound healing, easy bruising, and sometimes signs of arterial fragility. In addition, they show subtle signs of OI including blue sclerae, relatively short stature and osteopenia or fractures.
Recognition of this distinct phenotype is important for accurate genetic counselling, clinical management and surveillance, particularly in relation to the potential risk for vascular rupture associated with these mutations. Because these patients present clinical overlap with other EDS subtypes, biochemical collagen analysis is necessary to establish the correct diagnosis.
PMCID: PMC3662563  PMID: 23692737
Ehlers-Danlos syndrome; Osteogenesis Imperfecta; Type I collagen; Arterial fragility; Genotype; Phenotype
12.  Zebrafish Models for Ectopic Mineralization Disorders: Practical Issues from Morpholino Design to Post-Injection Observations 
Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and ZF. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization-related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos into early stage embryos. Validation of the morpholino needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining, and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization-related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of ZF models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification.
PMCID: PMC3669896  PMID: 23760765
zebrafish; embryos; morpholino; mineralization; osteogenic pathways
13.  GLUT10 is required for the development of the cardiovascular system and the notochord and connects mitochondrial function to TGFβ signaling 
Human Molecular Genetics  2011;21(6):1248-1259.
Growth factor signaling results in dramatic phenotypic changes in cells, which require commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member of the facilitative glucose transporter family, are associated with altered transforming growth factor-β (TGFβ) signaling in patients with arterial tortuosity syndrome (ATS). The objective of this work was to test whether SLC2A10/GLUT10 can serve as a link between TGFβ-related transcriptional regulation and metabolism during development. In zebrafish embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and blood flow, which was coupled with an incomplete and irregular vascular patterning. This was phenocopied by treatment with a small-molecule inhibitor of TGFβ receptor (tgfbr1/alk5). Array hybridization showed that the changes at the transcriptome level caused by the two treatments were highly correlated, revealing that a reduced tgfbr1 signaling is a key feature of ATS in early zebrafish development. Interestingly, a large proportion of the genes, which were specifically dysregulated after glut10 depletion gene and not by tgfbr1 inhibition, play a major role in mitochondrial function. Consistent with these results, slc2a10 morphants showed decreased respiration and reduced TGFβ reporter gene activity. Finally, co-injection of antisense morpholinos targeting slc2a10 and smad7 (a TGFβ inhibitor) resulted in a partial rescue of smad7 morphant phenotypes, suggesting scl2a10/glut10 functions downstream of smads. Taken together, glut10 is essential for cardiovascular development by facilitating both mitochondrial respiration and TGFβ signaling.
PMCID: PMC3284116  PMID: 22116938
14.  Twenty patients including 7 probands with autosomal dominant cutis laxa confirm clinical and molecular homogeneity 
Elastin gene mutations have been associated with a variety of phenotypes. Autosomal dominant cutis laxa (ADCL) is a rare disorder that presents with lax skin, typical facial characteristics, inguinal hernias, aortic root dilatation and pulmonary emphysema. In most patients, frameshift mutations are found in the 3’ region of the elastin gene (exons 30-34) which result in a C-terminally extended protein, though exceptions have been reported.
We clinically and molecularly characterized the thus far largest cohort of ADCL patients, consisting of 19 patients from six families and one sporadic patient.
Molecular analysis showed C-terminal frameshift mutations in exon 30, 32, and 34 of the elastin gene and identified a mutational hotspot in exon 32 (c.2262delA). This cohort confirms the previously reported clinical constellation of skin laxity (100%), inguinal hernias (51%), aortic root dilatation (55%) and emphysema (37%).
ADCL is a clinically and molecularly homogeneous disorder, but intra- and interfamilial variability in the severity of organ involvement needs to be taken into account. Regular cardiovascular and pulmonary evaluations are imperative in the clinical follow-up of these patients.
PMCID: PMC3599008  PMID: 23442826
Elastin; ELN; Autosomal dominant cutis laxa; Genotype; Phenotype
15.  Genes in thoracic aortic aneurysms/dissections - do they matter? 
PMCID: PMC3741818  PMID: 23977562
17.  Characterization of a distinct lethal arteriopathy syndrome in twenty-two infants associated with an identical, novel mutation in FBLN4 gene, confirms fibulin-4 as a critical determinant of human vascular elastogenesis 
Vascular elasticity is crucial for maintaining hemodynamics. Molecular mechanisms involved in human elastogenesis are incompletely understood. We describe a syndrome of lethal arteriopathy associated with a novel, identical mutation in the fibulin 4 gene (FBLN4) in a unique cohort of infants from South India.
Clinical characteristics, cardiovascular findings, outcomes and molecular genetics of twenty-two infants from a distinct population subgroup, presenting with characteristic arterial dilatation and tortuosity during the period August 2004 to June 2011 were studied.
Patients (11 males, 11 females) presented at median age of 1.5 months, belonging to unrelated families from identical ethno-geographical background; eight had a history of consanguinity. Cardiovascular features included aneurysmal dilatation, elongation, tortuosity and narrowing of the aorta, pulmonary artery and their branches. The phenotype included a variable combination of cutis laxa (52%), long philtrum-thin vermillion (90%), micrognathia (43%), hypertelorism (57%), prominent eyes (43%), sagging cheeks (43%), long slender digits (48%), and visible arterial pulsations (38%). Genetic studies revealed an identical c.608A > C (p. Asp203Ala) mutation in exon 7 of the FBLN4 gene in all 22 patients, homozygous in 21, and compound heterozygous in one patient with a p. Arg227Cys mutation in the same conserved cbEGF sequence. Homozygosity was lethal (17/21 died, median age 4 months). Isthmic hypoplasia (n = 9) correlated with early death (≤4 months).
A lethal, genetic disorder characterized by severe deformation of elastic arteries, was linked to novel mutations in the FBLN4 gene. While describing a hitherto unreported syndrome in this population subgroup, this study emphasizes the critical role of fibulin-4 in human elastogenesis.
PMCID: PMC3598868  PMID: 22943132
Arterial tortuosity; Fibulin-4 mutation; Aortic aneurysm; Vascular elasticity; Genetic vasculopathy; Mappila muslims; Founder effect; Cardiovascular imaging; Lethal mutation; Connective tissue disorder; Abnormal elastogenesis; Malabar
18.  New insights into the pathogenesis of autosomal dominant cutis laxa with report of five ELN mutations 
Human Mutation  2011;32(4):445-455.
Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema. We sequenced exons 28–34 of the ELN gene in 5 probands with ADCL features and found 5 de novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG (CL-3), c.2262delA (monozygotic twin CL-4 and CL-5) and c.2124del25 (CL-6). Four probands (CL-1, -2, -3, -6) presented with progressive aortic root dilatation. CL-2 and CL-3 also had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies showed stable mutant mRNA in all patients. Exon 32 skipping explains a milder phenotype in patients with exon 32 mutations. Mutant protein expression in fibroblast cultures impaired deposition of tropoelastin onto microfibril-containing fibers, and enhanced tropoelastin coacervation and globule formation leading to lower amounts of mature, insoluble elastin. Mutation-specific effects also included endoplasmic reticulum stress and increased apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming growth factor beta (TGFβ) signaling. We conclude that ADCL is a systemic disease with cardiovascular and pulmonary complications, associated with increased TGFβ signaling and mutation-specific differences in endoplasmic reticulum stress and apoptosis.
PMCID: PMC3383654  PMID: 21309044
ELN; CL; connective tissue; skin; aneurysm; emphysema
19.  Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum 
Cell Cycle  2011;10(11):1810-1820.
Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6-/- mice. Abcc6-/- mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.
PMCID: PMC3142464  PMID: 21597330
pseudoxanthoma elasticum; vitamin K; mineralization; Abcc6; mouse
20.  Mutations in FKBP10 Cause Recessive Osteogenesis Imperfecta and Bruck Syndrome 
Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179293  PMID: 20839288
21.  Histopathology of Pseudoxanthoma Elasticum and Related Disorders: Histological Hallmarks and Diagnostic Clues 
Scientifica  2012;2012:598262.
Among ectopic mineralization disorders, pseudoxanthoma elasticum (PXE)—a rare genodermatosis associated with ocular and cardiovascular manifestations—is considered a paradigm disease. The symptoms of PXE are the result of mineralization and fragmentation of elastic fibers, the exact pathophysiology of which is incompletely understood. Though molecular analysis of the causal gene, ABCC6, has a high mutation uptake, a skin biopsy has until now been considered the golden standard to confirm the clinical diagnosis. Although the histological hallmarks of PXE are rather specific, several other diseases—particularly those affecting the skin—can present with clinical and/or histological characteristics identical to or highly resemblant of PXE. In this paper, we will summarize the histopathological features of PXE together with those of disorders that are most frequently considered in the differential diagnosis of PXE.
PMCID: PMC3820553  PMID: 24278718
22.  Osteogenesis imperfecta: the audiological phenotype lacks correlation with the genotype 
Osteogenesis Imperfecta (OI) is a heritable connective tissue disorder mainly caused by mutations in the genes COL1A1 and COL1A2 and is associated with hearing loss in approximately half of the cases. The hearing impairment usually starts between the second and fourth decade of life as a conductive hearing loss, frequently evolving to mixed hearing loss thereafter. A minority of patients develop pure sensorineural hearing loss. The interindividual variability in the audiological characteristics of the hearing loss is unexplained.
With the purpose of evaluating inter- and intrafamilial variability, hearing was thorougly examined in 184 OI patients (type I: 154; type III: 4; type IV: 26), aged 3-89 years, with a mutation in either COL1A1 or COL1A2 and originating from 89 different families. Due to the adult onset of hearing loss in OI, correlations between the presence and/or characteristics of the hearing loss and the underlying mutation were investigated in a subsample of 114 OI patients from 64 different families who were older than 40 years of age or had developed hearing loss before the age of 40.
Hearing loss was diagnosed in 48.4% of the total sample of OI ears with increasing prevalence in the older age groups. The predominant type was a mixed hearing loss (27.5%). A minority presented a pure conductive (8.4%) or pure sensorineural (12.5%) loss. In the subsample of 114 OI subjects, no association was found between the nature of the mutation in COL1A1 or COL1A2 genes and the occurrence, type or severity of hearing loss. Relatives originating from the same family differed in audiological features, which may partially be attributed to their dissimilar age.
Our study confirms that hearing loss in OI shows a strong intrafamilial variability. Additional modifications in other genes are assumed to be responsible for the expression of hearing loss in OI.
PMCID: PMC3267664  PMID: 22206639
Osteogenesis Imperfecta; COL1A1; COL1A2; hearing loss; genotype-phenotype correlation
24.  The microRNA body map: dissecting microRNA function through integrative genomics 
Nucleic Acids Research  2011;39(20):e136.
While a growing body of evidence implicates regulatory miRNA modules in various aspects of human disease and development, insights into specific miRNA function remain limited. Here, we present an innovative approach to elucidate tissue-specific miRNA functions that goes beyond miRNA target prediction and expression correlation. This approach is based on a multi-level integration of corresponding miRNA and mRNA gene expression levels, miRNA target prediction, transcription factor target prediction and mechanistic models of gene network regulation. Predicted miRNA functions were either validated experimentally or compared to published data. The predicted miRNA functions are accessible in the miRNA bodymap, an interactive online compendium and mining tool of high-dimensional newly generated and published miRNA expression profiles. The miRNA bodymap enables prioritization of candidate miRNAs based on their expression pattern or functional annotation across tissue or disease subgroup. The miRNA bodymap project provides users with a single one-stop data-mining solution and has great potential to become a community resource.
PMCID: PMC3203610  PMID: 21835775

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