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author:("couched, Paul")
1.  Perturbation of specific pro-mineralizing signalling pathways in human and murine pseudoxanthoma elasticum 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1750-1172-9-66
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
2.  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.
doi:10.1371/journal.pone.0089749
PMCID: PMC3933654  PMID: 24587008
3.  Novel MYH11 and ACTA2 mutations reveal a role for enhanced TGFβ signaling in FTAAD 
International journal of cardiology  2011;165(2):314-321.
Background
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.
Conclusions
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.
doi:10.1016/j.ijcard.2011.08.079
PMCID: PMC3253210  PMID: 21937134
thoracic aortic aneurysm; myosin heavy chain 11; smooth muscle α-actin; TGFβ signaling
4.  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.
doi:10.1186/1750-1172-8-154
PMCID: PMC3850743  PMID: 24079343
Osteogenesis imperfecta; Type I collagen; OASIS; CREB3L1; Endoplasmic reticulum stress
5.  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.
doi:10.3389/fgene.2013.00074
PMCID: PMC3669896  PMID: 23760765
zebrafish; embryos; morpholino; mineralization; osteogenic pathways
6.  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.
doi:10.1093/hmg/ddr555
PMCID: PMC3284116  PMID: 22116938
7.  Twenty patients including 7 probands with autosomal dominant cutis laxa confirm clinical and molecular homogeneity 
Background
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.
Methods
We clinically and molecularly characterized the thus far largest cohort of ADCL patients, consisting of 19 patients from six families and one sporadic patient.
Results
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%).
Conclusion
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.
doi:10.1186/1750-1172-8-36
PMCID: PMC3599008  PMID: 23442826
Elastin; ELN; Autosomal dominant cutis laxa; Genotype; Phenotype
8.  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 
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1186/1750-1172-7-61
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
9.  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.
doi:10.1002/humu.21462
PMCID: PMC3383654  PMID: 21309044
ELN; CL; connective tissue; skin; aneurysm; emphysema
10.  Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform 
BMC Medical Genomics  2012;5:17.
Background
Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.
Results
In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.
Conclusions
We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.
doi:10.1186/1755-8794-5-17
PMCID: PMC3443074  PMID: 22607986
Deafness; Next generation sequencing; PCR based enrichment; Genetic diagnostics
11.  Osteogenesis imperfecta: the audiological phenotype lacks correlation with the genotype 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1750-1172-6-88
PMCID: PMC3267664  PMID: 22206639
Osteogenesis Imperfecta; COL1A1; COL1A2; hearing loss; genotype-phenotype correlation
12.  Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics 
PLoS ONE  2011;6(9):e25531.
Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics.
doi:10.1371/journal.pone.0025531
PMCID: PMC3184136  PMID: 21980484
14.  Stickler syndrome caused by COL2A1 mutations: genotype–phenotype correlation in a series of 100 patients 
Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome.
doi:10.1038/ejhg.2010.23
PMCID: PMC2987380  PMID: 20179744
COL2A1; Stickler syndrome; genotype–phenotype correlation; type II collagenopathies; splice site mutation
15.  Altered TGFβ signaling and cardiovascular manifestations in patients with autosomal recessive cutis laxa type I caused by fibulin-4 deficiency 
Fibulin-4 is a member of the fibulin family, a group of extracellular matrix proteins prominently expressed in medial layers of large veins and arteries. Involvement of the FBLN4 gene in cardiovascular pathology was shown in a murine model and in three patients affected with cutis laxa in association with systemic involvement. To elucidate the contribution of FBLN4 in human disease, we investigated two cohorts of patients. Direct sequencing of 17 patients with cutis laxa revealed no FBLN4 mutations. In a second group of 22 patients presenting with arterial tortuosity, stenosis and aneurysms, FBLN4 mutations were identified in three patients, two homozygous missense mutations (p.Glu126Lys and p.Ala397Thr) and compound heterozygosity for missense mutation p.Glu126Val and frameshift mutation c.577delC. Immunoblotting analysis showed a decreased amount of fibulin-4 protein in the fibroblast culture media of two patients, a finding sustained by diminished fibulin-4 in the extracellular matrix of the aortic wall on immunohistochemistry. pSmad2 and CTGF immunostaining of aortic and lung tissue revealed an increase in transforming growth factor (TGF)β signaling. This was confirmed by pSmad2 immunoblotting of fibroblast cultures. In conclusion, patients with recessive FBLN4 mutations are predominantly characterized by aortic aneurysms, arterial tortuosity and stenosis. This confirms the important role of fibulin-4 in vascular elastic fiber assembly. Furthermore, we provide the first evidence for the involvement of altered TGFβ signaling in the pathogenesis of FBLN4 mutations in humans.
doi:10.1038/ejhg.2010.45
PMCID: PMC2987390  PMID: 20389311
TGFβ; fibulin-4; cutis laxa; aortic aneurysm; arterial tortuosity
16.  Mutation detection in the ABCC6 gene and genotype–phenotype analysis in a large international case series affected by pseudoxanthoma elasticum 
Journal of Medical Genetics  2007;44(10):621-628.
Background
Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder with considerable phenotypic variability, mainly affects the eyes, skin and cardiovascular system, characterised by dystrophic mineralization of connective tissues. It is caused by mutations in the ABCC6 (ATP binding cassette family C member 6) gene, which encodes MRP6 (multidrug resistance‐associated protein 6).
Objective
To investigate the mutation spectrum of ABCC6 and possible genotype–phenotype correlations.
Methods
Mutation data were collected on an international case series of 270 patients with PXE (239 probands, 31 affected family members). A denaturing high‐performance liquid chromatography‐based assay was developed to screen for mutations in all 31 exons, eliminating pseudogene coamplification. In 134 patients with a known phenotype and both mutations identified, genotype–phenotype correlations were assessed.
Results
In total, 316 mutant alleles in ABCC6, including 39 novel mutations, were identified in 239 probands. Mutations were found to cluster in exons 24 and 28, corresponding to the second nucleotide‐binding fold and the last intracellular domain of the protein. Together with the recurrent R1141X and del23–29 mutations, these mutations accounted for 71.5% of the total individual mutations identified. Genotype–phenotype analysis failed to reveal a significant correlation between the types of mutations identified or their predicted effect on the expression of the protein and the age of onset and severity of the disease.
Conclusions
This study emphasises the principal role of ABCC6 mutations in the pathogenesis of PXE, but the reasons for phenotypic variability remain to be explored.
doi:10.1136/jmg.2007.051094
PMCID: PMC2597973  PMID: 17617515
pseudoxanthoma elasticum; ABC transporters; genotype–phenotype correlations; heritable skin diseases
17.  Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline 
BMC Bioinformatics  2010;11:269.
Background
Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions.
Results
We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data.
Conclusions
We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.
doi:10.1186/1471-2105-11-269
PMCID: PMC2880033  PMID: 20487544
18.  Clinical and mutation-type analysis from an international series of 198 probands with a pathogenic FBN1 exons 24-32 mutation 
Mutations in the FBN1 gene cause Marfan syndrome (MFS) and a wide range of overlapping phenotypes. The severe end of the spectrum is represented by neonatal MFS, the vast majority of probands carrying a mutation within exons 24-32. We previously showed that a mutation in exons 24-32 is predictive of a severe cardiovascular phenotype even in non-neonatal cases, and that mutations leading to premature truncation codons are under-represented in this region. To describe patients carrying a mutation in this so-called “neonatal” region, we studied the clinical and molecular characteristics of 198 probands with a mutation in exons 24-32 from a series of 1013 probands with a FBN1 mutation (20%). When comparing patients with mutations leading to a premature termination codon within exons 24-32 to patients with an in-frame mutation within the same region, a significantly higher probability of developing ectopia lentis and mitral insufficiency were found in the second group. Patients with a premature termination codon within exons 24-32 rarely displayed a neonatal or severe MFS presentation. We also found a higher probability of neonatal presentations associated with exon 25 mutations, as well as a higher probability of cardiovascular manifestations. A high phenotypic heterogeneity could be described for recurrent mutations, ranging from neonatal to classical MFS phenotype. In conclusion, even if the exon 24-32 location appears as a major cause of the severity of the phenotype in patients with a mutation in this region, other factors such as the type of mutation or modifier genes might also be relevant.
doi:10.1038/ejhg.2008.207
PMCID: PMC2734964  PMID: 19002209
Codon, Nonsense; DNA Mutational Analysis; Ectopia Lentis; genetics; Exons; genetics; Humans; Marfan Syndrome; genetics; Microfilament Proteins; genetics; metabolism; Mutation; Phenotype
19.  Genetic Screening of LCA in Belgium: Predominance of CEP290 and Identification of Potential Modifier Alleles in AHI1 of CEP290-related Phenotypes 
Human Mutation  2010;31(10):E1709-E1766.
Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early-onset retinal dystrophy (EORD) and 13 patients with Senior-Loken syndrome (SLS), LCA-Joubert syndrome (LCA-JS) or cerebello-oculo-renal syndrome (CORS) were included. Exhaustive re-inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290-related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290-related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290-related disease. © 2010 Wiley-Liss, Inc.
doi:10.1002/humu.21336
PMCID: PMC3048164  PMID: 20683928
LCA; CEP290; AHI1; modifier; genotype-phenotype correlation

Results 1-19 (19)