ZIC1 gene expression is controlled by DNA and histone methylation in mesenchymal proliferations

AbstractRNA and protein analysis revealed the consistent upregulation of the neural transcription factors ZIC1 and ZIC4 in desmoid tumors and other fibroproliferative disorders. The 5′ flanking region of the ZIC1 promoter was unmethylated in desmoid tumor fibroblasts, while a hypermethylated ZIC1 promoter was found in human and mouse cell lines not expressing the gene. In addition, expressing cells showed a H3K4me2 at the ZIC1 promoter, whereas non-expressing cells showed higher levels of H3K9me2 in the same region. To our knowledge, this is the first report describing ZIC1 expression in mesenchymal proliferations and a role for DNA methylation in the control of ZIC1 expression

The identification of new disease-associated genetic mechanisms in patients with mental retardation

Failure of normal brain development or functioning can lead to mental re tardation (MR), now often referred to as intellectual disability, charac terized by limitations in mental capabilities and adaptive skills. MR ha s a prevalence of 2-3% in the general population in developed countries. Though environmental factors significantly contribute to the appearance and severity of the disease, genetic factors are estimated to account f or 25-35%. Gene defects on the X chromosome (X-linked) have been conside red to be important contributors to MR and research has been mainly focu sed on this X-linked form of MR (XLMR). The etiology of XLMR is extremel y heterogeneous and requires the use of several molecular techniques to find them. This research project focuses on the identification of new XL MR-associated genetic factors or disease mechanisms. To this end, the de velopment and implementation of novel technologies were crucial. Mutations altering the coding sequence of a gene or one of its regulator y elements are the classical genetic defects associated with inherited h uman disorders. The standard molecular technique to track such changes i s via sequence analysis. Other, less common disease-associated mutations are chromosomal aberrations, which often are very informative. Whereas classical karyotyping allows rough estimation of the breakpoints of the chromosomal aberration, walking FISH can delineate the breakpoint region to a few tens of kb. Genome browsers are then consulted to check whethe r a gene could be interrupted by the breakpoint. Subsequently, altered e xpression of this candidate MR gene can be analyzed in patient derived c ells, which could explain the patient s phenotype. We characterized the breakpoints of a translocation t(X;9)(q13.2;p24) present in a severely d evelopmentally retarded female patient. The breakpoint on the X chromoso me was found to disrupt the MCT8 gene. Moreover, X-inactivation studi es revealed that the normal X chromosome was always inactivated in her c ells suggesting the lack of MCT8 expression, which was confirmed in t he patient s fibroblasts (part 1). As such, this patient represents t he first female with the Allen-Herndon-Dudley syndrome (AHDS). Up to now, 82 XLMR genes have been described but the mutation frequency of each gene in the XLMR population is very low (0.1-1%). The novel tech nology of array-CGH allowed the identification of submicroscopic genomic gains and losses (>5 Mb to 10 kb), which added another layer of disease -causing mutations in XLMR. The resolution of array-CGH correlates with the type and number of probes spotted onto the array: the initial resolu tion obtained with large-insert BAC/PAC clones spaced every 1 Mb was sig nificantly lower than the currently used oligonucleotides arrays spaced every 100 bp. We developed a full-tiling X chromosome-specific BAC array (X-array) wit h a theoretical resolution of 80 kb (part 2). The X-array was validat ed by hybridisation of DNA from patients with known X-chromosomal imbala nces of different sizes. All clones within the aberration (deletion as w ell as duplication) were easily detected, and the borders of the imbalan ce could be readily delineated demonstrating the usefulness of our X-arr ay. After validation, the array was used to screen a population of 108 patie nts with idiopathic MR and 15 genomic aberrations were found in 14 patie nts (13%); amongst these are 2 deletions and 13 duplications with variab le length (0.1-2.7 Mb) (part 2). To determine whether an aberration o n the X chromosome can be considered causal, we proposed the following c riteria: de novo aberration, segregation of the aberration in the family with the disease, absent in control individuals and nonrandom X-i nactivation in asymptomatic female carriers. Also, the presence of a kno wn (N)SXLMR gene can be a valuable indication. As such the aberration wa s suggested to be causal for the MR phenotype in 5 patients (4.6%). From this screening effort it was hypothesized that not only deletion, b ut also duplication of dosage-sensitive (N)SXLMR genes can result in XLM R. To test this hypothesis, we focused on the duplication of MECP2 , which was identified in four unrelated families. Via mRNA expression a nalysis we demonstrated a 2-fold higher expression of MECP2 in EBV -PBLs of patients with the duplication when compared to controls. Notabl y, it was reported that slight overexpression of MECP2 in Mecp2 -null mice resulted in neurological abnormalities, which underscores the biological relevance of our finding. Also, this dosage-sensitive rol e of MeCP2 in neuronal plasticity and function was observed in brain sli ces of patients with neurological abnormalities. As such, we defined gen e duplication of known (N)SXLMR genes as a new disease mechanism in XLMR (part 3). Several other causal duplications were later described by our group. The discovery of causal genomic duplications and deletions in disease in troduced the concept of genomic disorders, which are characterized by th e quantitative alteration of a dosage sensitive gene or genes via genomi c rearrangements. Since several unrelated patients often harbor a simila r genomic rearrangement, the question arises whether there is a common m echanism driving these rearrangements. We tackled this question via a st udy of the rather frequent nonrecurrent MECP2 duplication events. Bre akpoint mapping, in silico breakpoint analysis in 16 patients and seq uencing of the breakpoints in three patients revealed that the distinct complex genomic architecture at Xq28 can trigger the common NAHR and NHE J mechanisms but can also activate a combination of alternative repair p rocesses to restore genomic integrity, yielding either a simple or compl ex rearrangement (part 4). From this research project it is clear that a wide variety of molecular techniques is used to unravel the genetic causes underlying XLMR. Despit e extensive mutation screening via positional cloning, sequence analysis and array-CGH, the causal factor remains to be determined in >50% of th e XLMR patients. With the recent development of the high-throughput deep sequencing technologies, identification of additional genetic factors w ill speed up the mutation detection rate. Molecular passports will be ge nerated at base pair resolution; not only at the genomic but also at the epigenomic level because there is growing evidence for the involvement of the epigenome in neurological diseases and cognition. Large amounts o f raw data will be generated and it will be crucial to validate or compl ement those with accurate biological and functional data. In addition, o ther disease-associated mechanisms are hunted for and great efforts are currently being made to understand the involvement of regulatory element s and noncoding RNA s, or to investigate the importance of higher-order chromatin structure in normal development and disease.status: publishe

X-linked mental retardation and epigenetics

The search for the genetic defects in constitutional diseases has so far been restricted to direct methods for the identification of genetic mutations in the patients' genome. Traditional methods such as karyotyping, FISH, mutation screening, positional cloning and CGH, have been complemented with newer methods including array-CGH and PCR-based approaches (MLPA, qPCR). These methods have revealed a high number of genetic or genomic aberrations that result in an altered expression or reduced functional activity of key proteins. For a significant percentage of patients with congenital disease however, the underlying cause has not been resolved strongly suggesting that yet other mechanisms could play important roles in their etiology. Alterations of the 'native' epigenetic imprint might constitute such a novel mechanism. Epigenetics, heritable changes that do not rely on the nucleotide sequence, has already been shown to play a determining role in embryonic development, X-inactivation, and cell differentiation in mammals. Recent progress in the development of techniques to study these processes on full genome scale has stimulated researchers to investigate the role of epigenetic modifications in cancer as well as in constitutional diseases. We will focus on mental impairment because of the growing evidence for the contribution of epigenetics in memory formation and cognition. Disturbance of the epigenetic profile due to direct alterations at genomic regions, or failure of the epigenetic machinery due to genetic mutations in one of its components, has been demonstrated in cognitive derangements in a number of neurological disorders now. It is therefore tempting to speculate that the cognitive deficit in a significant percentage of patients with unexplained mental retardation results from epigenetic modifications.status: publishe

Challenges in molecular diagnosis of X-linked Intellectual disability

BACKGROUND: Intellectual disability (ID) affects 1-3% of the Western population and is heterogeneous in origin. Mutations in X-linked genes represent 5-10% of ID in males. Fragile X syndrome, due to the silencing of the FMR1 gene, is the most common form of ID, with a prevalence of around 1:5000 males. Females are usually non- or mildly affected carriers, and in a few rare cases, the only gender affected. Array comparative genome hybridization (aCGH) and next-generation sequencing (NGS) have dramatically changed the nature of human genome analysis leading to the identification of new X-linked intellectual disability syndromes and disease-causing genes. SOURCES OF DATA: Original papers, reviews, guidelines and experiences of the diagnostic laboratories. AREAS OF AGREEMENT: Family history and clinical examination still are essential to choose the appropriate diagnostic tests, including, a disease-specific genetic test, aCGH or FMR1 molecular analysis. If negative, NGS approaches like well-defined gene panels, whole exome, or even whole genome sequencing, are increasingly being used, improving diagnostics and leading to the identification of novel disease mechanisms. AREAS OF CONTROVERSY: The main challenge in the era of NGS is filtering and interpretation of the data generated by the analysis of a single individual. In X-linked cases, assessing pathogenicity is particularly challenging, even more when the variant is found to be inherited from a healthy carrier mother or when a heterozygous X-linked mutation is found in an impaired female. GROWING POINTS: At present, variant interpretation remains a challenging task, especially in X-linked disorders. We review the main difficulties and propose a comprehensive overview that might aid in variant interpretation. Establishing a genetic diagnosis facilitates counseling and allows better delineation of clinical phenotypes. AREAS TIMELY FOR DEVELOPING RESEARCH: To improve variant interpretation, there is need to refine in silico predictions with specific criteria for each gene, and to develop cost-effective functional tools, which can be easily transferred to diagnostics.status: publishe

Detection and validation of copy number variation in X-linked mental retardation

Studies to identify the genetic defects associated with X-linked mental retardation (XLMR) in males have revealed tens of genes important for normal brain development and cognitive functioning in men. Despite extensive efforts in breakpoint cloning of chromosomal rearrangements and mutation screening of candidate genes on the X chromosome, still many XLMR families and sporadic cases remain unsolved. It is now clear that submicroscopic copy number changes on the X chromosome can explain about 5% of these idiopathic cases. Interestingly, beside gene deletions, an increase in gene dosage due to genomic duplications seems to contribute to causality more often than expected. Since larger duplications on the X chromosome are tolerated compared to deletions, they often harbour more than one gene hampering the identification of the causal gene. In contrast to copy number variations (CNVs) on autosomes, most disease-associated CNVs on the X chromosome in males are inherited from their mothers who normally do not present with any clinical symptoms due to non-random X inactivation. Here, we review the different methods applied to study copy number alterations on the X chromosome in patients with cognitive impairment, discuss those CNVs that are associated with disease and elaborate on the genes and mechanisms involved. At the end, we will resume in vivo assays to study the relation of CNVs on the X chromosome and mental disability.status: publishe

Two cases of non-alcoholic fatty liver disease caused by biallelic ABHD5 mutations

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Genotype-phenotype correlations of UBA2 mutations in patients with ectrodactyly

Interstitial 19q13.11 deletions are associated with ectrodactyly, which has recently been linked to loss-of-function of the UBA2 gene. We report a boy with a de novo frameshift mutation in UBA2 (c.612delA (p.(Glu205Lysfs*63)), presenting with ectrodactyly of the feet associated with learning difficulties and minor physical anomalies. We review genotype-phenotype correlations in patients with chromosomal 19q13.11 microdeletions compared to those with intragenic UBA2 mutations.status: publishe

Congenital heart defects in a novel recurrent 22q11.2 deletion harboring the genes CRKL and MAPK1

The proximal region of the long arm of chromosome 22 is rich in low copy repeats (LCR). Non-allelic homologous recombination (NAHR) between these substrates explains the high prevalence of recurrent rearrangements within this region. We have performed array comparative genomic hybridization in a normally developing girl with growth delay, microcephaly, and truncus arteriosus, and have identified a novel recurrent 22q11 deletion that spans LCR22-4 and partially affects the common 22q11.2 deletion syndrome and the distal 22q11 deletion syndrome. This deletion is atypical as it did not occur by NAHR between any of the major LCRs found on 22q11.2. However, the breakpoint containing regions coincide with highly homologous regions. An identical imbalance was reported previously in a patient with striking phenotypic similarity. Computational gene prioritization methods and biological evidence denote the genes CRKL and MAPK1 as the highest ranking candidates for causing congenital heart disease within the deleted region. © 2012 Wiley Periodicals, Inc.status: publishe