http://repub.eur.nl/res/aut/11867/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/28696/ 2010-09-14T00:00:00Z Background: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. Methodology: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. Principal Findings: Our previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. Conclusions: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE. http://repub.eur.nl/res/pub/20895/ 2010-09-01T00:00:00Z Refractive errors are the most common ocular disorders worldwide and may lead to blindness. Although this trait is highly heritable, identification of susceptibility genes has been challenging. We conducted a genome-wide association study for refractive error in 5,328 individuals from a Dutch population-based study with replication in four independent cohorts (combined 10,280 individuals in the replication stage). We identified a significant association at chromosome 15q14 (rs634990, P = 2.21 × 10−14). The odds ratio of myopia compared to hyperopia for the minor allele (minor allele frequency = 0.47) was 1.41 (95% CI 1.16–1.70) for individuals heterozygous for the allele and 1.83 (95% CI 1.42–2.36) for individuals homozygous for the allele. The associated locus is near two genes that are expressed in the retina, GJD2 and ACTC1, and appears to harbor regulatory elements which may influence transcription of these genes. Our data suggest that common variants at 15q14 influence susceptibility for refractive errors in the general population. http://repub.eur.nl/res/pub/20901/ 2010-09-01T00:00:00Z Refractive errors are the most common ocular disorders worldwide and may lead to blindness. Although this trait is highly heritable, identification of susceptibility genes has been challenging. We conducted a genome-wide association study for refractive error in 5,328 individuals from a Dutch population-based study with replication in four independent cohorts (combined 10,280 individuals in the replication stage). We identified a significant association at chromosome 15q14 (rs634990, P = 2.21 × 10−14). The odds ratio of myopia compared to hyperopia for the minor allele (minor allele frequency = 0.47) was 1.41 (95% CI 1.16–1.70) for individuals heterozygous for the allele and 1.83 (95% CI 1.42–2.36) for individuals homozygous for the allele. The associated locus is near two genes that are expressed in the retina, GJD2 and ACTC1, and appears to harbor regulatory elements which may influence transcription of these genes. Our data suggest that common variants at 15q14 influence susceptibility for refractive errors in the general population. http://repub.eur.nl/res/pub/28249/ 2010-08-01T00:00:00Z Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12-13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels. http://repub.eur.nl/res/pub/28295/ 2010-06-01T00:00:00Z Genetic alterations in the alpha-synuclein (SNCA) gene have been implicated in Parkinson Disease (PD), including point mutations, gene multiplications, and sequence variations within the promoter. Such alterations may be involved in pathology through structural changes or overexpression of the protein leading to protein aggregation, as well as through impaired gene expression. It is, therefore, of importance to specify the parameters that regulate SNCA expression in its normal and mutated state. We studied the expression of SNCA alleles in a lymphoblastoid cell line and in the blood cells of a patient heterozygous for p.Ala53Thr, the first mutation to be implicated in PD pathogenesis. Here, we provide evidence that: (1) SNCA shows monoallelic expression in this patient, (2) epigenetic silencing of the mutated allele involves histone modifications but not DNA methylation, and (3) steady-state mRNA levels deriving from the normal SNCA allele in this patient exceed those of the two normal SNCA alleles combined, in matching, control individuals. An imbalanced SNCA expression in this patient is thus documented, with silencing of the p.Ala53Thr allele and upregulation of the wild-type-allele. This phenomenon is demonstrated for a first time in the SNCA gene, and may have important implications for PD pathogenesis. http://repub.eur.nl/res/pub/28556/ 2010-06-01T00:00:00Z The combination of megalencephaly, perisylvian polymicrogyria, polydactyly and hydrocephalus (MPPH) is a rare syndrome of unknown cause. We observed two first cousins affected by an MPPH-like phenotype with a submicroscopic chromosome 5q35 deletion as a result of an unbalanced der(5)t(5;20)(q35.2;q13.3) translocation, including the NSD1 Sotos syndrome locus. We describe the phenotype and the deletion breakpoints of the two MPPH-like patients and compare these with five unrelated MPPH and Sotos patients harboring a 5q35 microdeletion. Mapping of the breakpoints in the two cousins was performed by MLPA, FISH, high density SNP-arrays and Q-PCR for the 5q35 deletion and 20q13 duplication. The 5q35 deletion area of the two cousins almost completely overlaps with earlier described patients with an atypical Sotos microdeletion, except for the DRD1 gene. The five unrelated MPPH patients neither showed submicroscopic chromosomal aberrations nor DRD1 mutations. We reviewed the brain MRI of 10 Sotos patients and did not detect polymicrogyria in any of them. In our two cousins, the MPPH-like phenotype is probably caused by the contribution of genes on both chromosome 5q35 and 20q13. Some patients with MPPH may harbor a submicroscopic chromosomal aberration and therefore high-resolution array analysis should be part of the diagnostic workup. http://repub.eur.nl/res/pub/28558/ 2010-04-01T00:00:00Z Malformations of cerebral cortical development, in particular periventricular nodular heterotopia(PNH), and distal transverse limb deficiency have been reported as associated congenital anomalies. Patients with PNH and transverse limb deficiency can be classified as having amniotic band sequence or Adams - Oliver syndrome (AOS). Controversy exists whether these should be considered separate entities. In some AOS patients, autosomal recessive inheritance has been shown, but in most patients causes are unknown, and both environmental and genetic factors have been implicated. We present three patients with PNH and distal transverse limb deficiency to support the hypothesis that these should be considered part of one group of disorders, and highlight the variable severity of the clinical and neuroradiological phenotype. Chromosome abnormalities were excluded by copy number analysis on 250K SNP microarray data.Research done on limb deficiency as on PNH caused by mutations in known genes, suggests the involvement of vascular developmental pathways. The combination of limb deficiency and PNH may have a common causative mechanism. Recognition and grouping of patients with this combination of abnormalities will help elucidating the cause. http://repub.eur.nl/res/pub/23079/ 2010-02-01T00:00:00Z Abstract: Attention deficit hyperactivity disorder (ADHD) is a common neuropsychiatric disorder. Genetics has an important role in the aetiology of this disease. In this study, we describe the clinical findings in a Dutch family with eight patients suffering from ADHD, in whom five had at least one other psychiatric disorder. We performed a genome-wide (parametric and nonparametric) affected-only linkage analysis. Two genomic regions on chromosomes 7 and 14 showed an excess of allele sharing among the definitely affected members of the family with suggestive LOD scores (2.1 and 2.08). Nonparametric linkage analyses (NPL) yielded a maxNPL of 2.92 (P=0.001) for marker D7S502 and a maxNPL score of 2.56 (P=0.003) for marker D14S275. We confirmed that all patients share the same haplotype in each region of 7p15.1-q31.33 and 14q11.2-q22.3. Interestingly, both loci have been reported before in Dutch (affected sib pairs) and German (extended families) ADHD linkage studies. Hopefully, the genome-wide association studies in ADHD will help to highlight specific polymorphisms and genes within the broad areas detected by our, as well as other, linkage studies. http://repub.eur.nl/res/pub/25344/ 2009-08-27T00:00:00Z Background. Complex chromosomal rearrangements (CCR) are rare cytogenetic findings that are difficult to karyotype by conventional cytogenetic analysis partially because of the relative low resolution of this technique. High resolution genotyping is necessary in order to identify cryptic imbalances, for instance near the multiple breakpoints, to explain the abnormal phenotype in these patients. We applied several molecular techniques to elucidate the complexity of the CCRs of two adult patients with abnormal phenotypes. Results. Multicolour fluorescence in situ hybridization (M-FISH) showed that in patient 1 the chromosomes 1, 10, 15 and 18 were involved in the rearrangement whereas for patient 2 the chromosomes 5, 9, 11 and 13 were involved. A 250 k Nsp1 SNP-array analysis uncovered a deletion in chromosome region 10p13 for patient 1, harbouring 17 genes, while patient 2 showed no pathogenic gains or losses. Additional FISH analysis with locus specific BAC-probes was performed, leading to the identification of cryptic interstitial structural rearrangements in both patients. Conclusion. Application of M-FISH and SNP-array analysis to apparently balanced CCRs is useful to delineate the complex chromosomal rearrangement in detail. However, it does not always identify cryptic imbalances as an explanation for the abnormal phenotype in patients with a CCR. http://repub.eur.nl/res/pub/24252/ 2009-07-10T00:00:00Z Cerebral palsy due to perinatal injury to cerebral white matter is usually not caused by genetic mutations, but by ischemia and/or inflammation. Here, we describe an autosomal-recessive type of tetraplegic cerebral palsy with mental retardation, reduction of cerebral white matter, and atrophy of the cerebellum in an inbred sibship. The phenotype was recorded and evolution followed for over 20 years. Brain lesions were studied by diffusion tensor MR tractography. Homozygosity mapping with SNPs was performed for identification of the chromosomal locus for the disease. In the 14 Mb candidate region on chromosome 7q22, RNA expression profiling was used for selecting among the 203 genes in the area. In postmortem brain tissue available from one patient, histology and immunohistochemistry were performed. Disease course and imaging were mostly reminiscent of hypoxic-ischemic tetraplegic cerebral palsy, with neuroaxonal degeneration and white matter loss. In all five patients, a donor splice site pathogenic mutation in intron 14 of the AP4M1 gene (c.1137+1G→T), was identified. AP4M1, encoding for the μ subunit of the adaptor protein complex-4, is involved in intracellular trafficking of glutamate receptors. Aberrant GluRδ2 glutamate receptor localization and dendritic spine morphology were observed in the postmortem brain specimen. This disease entity, which we refer to as congenital spastic tetraplegia (CST), is therefore a genetic model for congenital cerebral palsy with evidence for neuroaxonal damage and glutamate receptor abnormality, mimicking perinatally acquired hypoxic-ischemic white matter injury. http://repub.eur.nl/res/pub/24939/ 2009-04-20T00:00:00Z Background: To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63-78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software. Results: In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover. Conclusion: Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for candidate retinal disease gene identification or gene dose-dependent therapeutic studies. http://repub.eur.nl/res/pub/9079/ 1999-01-01T00:00:00Z In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin. http://repub.eur.nl/res/pub/13739/ 1994-02-23T00:00:00Z