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J T den Dunnen

Publications and source records attributed to J T den Dunnen.

At least 19 recordsLinked to original sources
PubMed2006

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

Cohort Studies↗
PubMed2006

Copy number variation in the genome; the human DMD gene as an example.

Recent developments have yielded new technologies that have greatly simplified the detection of deletions and duplications, i.e., copy number variants (CNVs). These technologies can be used to screen for CNVs in and around specific genomic regions, as well as genome-wide. Several genome-wide studies have demonstrated that CNV in the human genome is widespread and may include millions of nucleotides. One of the questions that emerge is which sequences, structures and/or processes are involved in their generation. Using as an example the human DMD gene, mutations in which cause Duchenne and Becker muscular dystrophy, we review the current data, determine the deletion and duplication profile across the gene and summarize the information that has been collected regarding their origin. In addition we discuss the methods most frequently used for their detection, in particular MAPH and MLPA.

Alleles↗
PubMed2005

Common pathological mechanisms in mouse models for muscular dystrophies.

Duchenne/Becker and limb-girdle muscular dystrophies share clinical symptoms like muscle weakness and wasting but differ in clinical presentation and severity. To get a closer view on the differentiating molecular events responsible for the muscular dystrophies, we have carried out a comparative gene expression profiling of hindlimb muscles of the following mouse models: dystrophin-deficient (mdx, mdx(3cv)), sarcoglycan-deficient (Sgca null, Sgcb null, Sgcg null, Sgcd null), dysferlin-deficient (Dysf null, SJL(Dysf)), sarcospan-deficient (Sspn null), and wild-type (C57Bl/6, C57Bl/10) mice. The expression profiles clearly discriminated between severely affected (dystrophinopathies and sarcoglycanopathies) and mildly or nonaffected models (dysferlinopathies, sarcospan-deficiency, wild-type). Dystrophin-deficient and sarcoglycan-deficient profiles were remarkably similar, sharing inflammatory and structural remodeling processes. These processes were also ongoing in dysferlin-deficient animals, albeit at lower levels, in agreement with the later age of onset of this muscular dystrophy. The inflammatory proteins Spp1 and S100a9 were up-regulated in all models, including sarcospan-deficient mice, which points, for the first time, at a subtle phenotype for Sspn null mice. In conclusion, we identified biomarker genes for which expression correlates with the severity of the disease, which can be used for monitoring disease progression. This comparative study is an integrating step toward the development of an expression profiling-based diagnostic approach for muscular dystrophies in humans.

Animals↗
PubMed2005

Muscle regeneration in dystrophin-deficient mdx mice studied by gene expression profiling.

BACKGROUND: Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is lethal. In contrast, dystrophin-deficient mdx mice recover due to effective regeneration of affected muscle tissue. To characterize the molecular processes associated with regeneration, we compared gene expression levels in hindlimb muscle tissue of mdx and control mice at 9 timepoints, ranging from 1-20 weeks of age. RESULTS: Out of 7776 genes, 1735 were differentially expressed between mdx and control muscle at at least one timepoint (p < 0.05 after Bonferroni correction). We found that genes coding for components of the dystrophin-associated glycoprotein complex are generally downregulated in the mdx mouse. Based on functional characteristics such as membrane localization, signal transduction, and transcriptional activation, 166 differentially expressed genes with possible functions in regeneration were analyzed in more detail. The majority of these genes peak at the age of 8 weeks, where the regeneration activity is maximal. The following pathways are activated, as shown by upregulation of multiple members per signalling pathway: the Notch-Delta pathway that plays a role in the activation of satellite cells, and the Bmp15 and Neuregulin 3 signalling pathways that may regulate proliferation and differentiation of satellite cells. In DMD patients, only few of the identified regeneration-associated genes were found activated, indicating less efficient regeneration processes in humans. CONCLUSION: Based on the observed expression profiles, we describe a model for muscle regeneration in mdx mice, which may provide new leads for development of DMD therapies based on the improvement of muscle regeneration efficacy.

Adolescent↗
PubMed2005

Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification.

BACKGROUND: Approximately 80% of the alpha- and 10% of the beta-thalassaemias are caused by genomic deletions involving the alpha- and beta-globin gene clusters on chromosomes 16p13.3 and 11p15.5, respectively. Gap-PCR, Southern blot analysis, and fluorescent in situ hybridisation are commonly used to identify these deletions; however, many deletions go undetected using conventional techniques. METHODS: Patient samples for which no abnormalities had been found using conventional DNA techniques were analysed by a three colour multiplex ligation-dependent probe amplification assay. Two sets of 35 and 50 probes, covering a region of 700 kb of the alpha- and 500 kb of the beta-globin gene cluster, respectively, were designed to detect rearrangements in the alpha- and beta-globin gene clusters. RESULTS: In 19 out of 38 patient samples, we found 11 different alpha-thalassaemia deletions, six of which were not previously described. Two novel deletions leaving the alpha-globin gene cluster intact were found to cause a complete downregulation of the downstream alpha-genes. Similarly, 31 out of 51 patient samples were found to carry 10 different deletions involving the beta-globin gene cluster, three of which were not previously described. One involves the deletion of the locus control region leaving the beta-globin gene cluster intact. CONCLUSIONS: These deletions, which are not easily detected by conventional techniques, may have clinical implications during pregnancy ranging from mild to life threatening microcytic haemolytic anaemia in neonates. The approach as described here is a rapid and sensitive method for high resolution analysis of the globin gene clusters and for any region of the genome.

Blotting, Southern↗
PubMed2005

Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort.

Mutations in the DMD gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Readily available clinical tests detect only deletions of one exon or greater, which are found in approximately 60% of cases. Mutational analysis of other types of DMD mutations, such as premature stop codons and small frameshifting insertions or deletions, has historically been hampered by the large size of the gene. We have recently reported a method that allows the rapid and economical sequencing of the entire coding region of the DMD gene, and that is more sensitive than methods based on single-strand conformational polymorphism (SSCP) screening or other preliminary screening steps. Here we use single condition amplification/internal primer (SCAIP) sequencing analysis, in combination with multiplex amplifiable probe hybridization (MAPH) analysis of duplications, to report the frequency of mutations in a large cohort of unselected dystrophinopathy patients from a single clinic. Our results indicate that 7% of dystrophinopathy patients do not have coding region mutations, suggesting that intronic mutations are not uncommon. The availability of rapid and thorough mutation analysis from peripheral blood samples, along with an improved estimate of the percentage of non-coding region mutations, will be of benefit for improved genetic counseling and in identification of cohorts for clinical trials.

Codon, Nonsense↗
PubMed2004

Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells.

As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 2'-O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholino-induced skipping levels were only 5-6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficiencies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping.

Base Sequence↗
PubMed2004

Genomic imbalances in mental retardation.

INTRODUCTION: It has been estimated that cytogenetically visible rearrangements are present in approximately 1% of newborns. These chromosomal changes can cause a wide range of deleterious developmental effects, including mental retardation (MR). It is assumed that many other cases exist where the cause is a submicroscopic deletion or duplication. To facilitate the detection of such cases, different techniques have been developed, which have differing efficiency as to the number of loci and patients that can be tested. METHODS: We implemented multiplex amplifiable probe hybridisation (MAPH) to test areas known to be rearranged in MR patients (for example, subtelomeric/pericentromeric regions and those affected in microdeletion syndromes) and to look for new regions that might be related to MR. RESULTS: In this study, over 30 000 screens for duplications and deletions were carried out; 162 different loci tested in each of 188 developmentally delayed patients. The analysis resulted in the detection of 19 rearrangements, of which approximately 65% would not have been detected by conventional cytogenetic analysis. A significant fraction (46%) of the rearrangements found were interstitial, despite the fact that only a limited number of these loci have so far been tested. DISCUSSION: Our results strengthen the arguments for whole genome screening within this population, as it can be assumed that many more interstitial rearrangements would be detected. The strengths of MAPH for this analysis are the simplicity, the high throughput potential, and the high resolution of analysis. This combination should help in the future identification of the specific genes that are responsible for MR.

Abnormalities, Multiple↗
PubMed2003

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

Amino Acid Sequence↗
PubMed2002

[From gene to disease; the dystrophin gene involved in Duchenne and Becker muscular dystrophy].

Duchenne and Becker muscular dystrophy (DMD and BMD) are progressive disorders, which almost exclusively affect males. DMD is the more severe type with an onset at 2-3 years of age. Patients become wheelchair-bound before the age of 13 and often die due to cardiac arrest or respiratory insufficiency. BMD, a more varying phenotype which may overlap with limb girdle muscular dystrophy (LGMD), has a less severe muscle weakness which starts later than in DMD patients. DMD carriers may show some muscle weakness. The dystrophin gene (2.4 Mb), known to be involved in DMD/BMD, codes for a 427 kilodalton muscle-specific protein named dystrophin as well as several tissue-specific isoforms. Dystrophin, as part of a membrane-bound complex of proteins, connects the cytoskeleton of the muscle cell to the extracellular matrix. Since 1985, when highly reliable carrier detection and prenatal diagnosis at the DNA level became possible, over 250 prenatal tests have been performed. Molecular genetic analysis, highlighted a phenomenon called germinal mosaicism, which explains the recurrence of de novo mutations and led to the discovery of the so-called reading-frame rule, which helps to discriminate between DMD and BMD. Fifteen years after the discovery of the dystrophin gene, mutations can be detected in 95% of the patients, while the remaining 5% are still hiding within this very large gene.

Chromosome Mapping↗
PubMed2001

Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells.

Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.

Alleles↗
PubMed2001

WHSC1L1, on human chromosome 8p11.2, closely resembles WHSC1 and maps to a duplicated region shared with 4p16.3.

We have identified and characterized a gene (60% on protein level) and a pseudogene (93% on DNA level) that show high similarity to the Wolf-Hirschhorn syndrome candidate gene-1 (WHSC1). These genes, WHSC1L1 and WHSC1L2P, map to human chromosomes 8p11.2 and 17q21, respectively. WHSC1L1 is ubiquitously expressed and, like WHSC1, generates two major transcripts, a short (s-type) and a long (l-type). The WHSC1L1 l-type transcript encodes a 1437-amino-acid protein containing 2 PWWP (proline-trypto-phan-proline-tryptophan) domains, 5 PHD (plant-home-domain)-type zinc finger motifs, a SAC (SET-associated Cys-rich) domain, and a SET (Suppressor of Variegation, Enhancer of Zeste and Trithorax) domain. The s-type transcript encodes a protein of 645 amino acids containing a PWWP domain only. WHSC1L2P is an unexpressed, intronless pseudogene of a WHSC1L1 s-type transcript. The 8p11.2 region around WHSC1L1 contains a set of genes including TACC1, FGFR1, LETM2, and WHSC1L1, which seems to be derived from a recent duplication involving 4p16.3 where a similar set of genes is located. Rearrangements of 8p are frequently found in human cancer, including breast cancer. These characteristics indicate that WHSC1L1 might have a role in embryonic development and, when disregulated, in cancer development.

Carrier Proteins↗
PubMed2001

Nomenclature for the description of human sequence variations.

A nomenclature system has recently been suggested for the description of changes (mutations and polymorphisms) in DNA and protein sequences. These nomenclature recommendations have now been largely accepted. However, current rules do not yet cover all types of mutations, nor do they cover more complex mutations. This document lists the existing recommendations and summarizes suggestions for the description of additional, more complex changes. Another version of this paper has been published in Hum Mut 15:7-12, 2000.

Amino Acid Substitution↗
PubMed2001

A partial BRCA1 sequence homology mapping to 4q28.

Using a BRCA1 cDNA probe in Southern analysis, we detected a sequence of 348 bp on 4q28 that is homologous to the 3' end of BRCA1. A 28-kb sequence contig has been assembled spanning the homologous region, which we designated BRCA1-h. An open reading frame was identified encoding a sequence of 82 amino acids; 22 of the last 23 amino acids are identical to the last 23 residues of BRCA1. BLAST-searches, RT-PCR and RACE-experiments have been unable to provide evidence that BRCA1-h is part of an expressed gene.

Amino Acid Sequence↗
PubMed2000

The PWWP domain: a potential protein-protein interaction domain in nuclear proteins influencing differentiation?

Upon characterization of WHSC1, a gene mapping to the Wolf-Hirschhorn syndrome critical region and at its C-terminus similar to the Drosophila ASH1/trithorax group proteins, we identified a novel protein domain designated PWWP domain. To gain insight into its structure, evolutionary conservation and its potential functional role, we performed database searches to identify other PWWP domain-containing proteins. We retrieved 39 proteins, and a multiple alignment shows that the domain spans some 70 amino acids. It is present in proteins of nuclear origin and plays a role in cell growth and differentiation. Due to its position, the composition of amino acids close to the PWWP motif and the pattern of other domains present, we hypothesize that the domain is involved in protein-protein interactions.

Amino Acid Motifs↗
PubMed2000

Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion.

Consistent gene mutation nomenclature is essential for efficient and accurate reporting, testing, and curation of the growing number of disease mutations and useful polymorphisms being discovered in the human genome. While a codified mutation nomenclature system for simple DNA lesions has now been adopted broadly by the medical genetics community, it is inherently difficult to represent complex mutations in a unified manner. In this article, suggestions are presented for reporting just such complex mutations.

Base Sequence↗
PubMed2000

Dystrophin nonsense mutation induces different levels of exon 29 skipping and leads to variable phenotypes within one BMD family.

Within one X-linked muscular dystrophy family, different phenotypes for three males occurred: (1) a severely affected Becker patient with cardiomyopathy, (2) a mildly affected Becker patient, and (3) an apparently healthy male with elevated serum CK levels. In the muscle biopsy specimen of patient2 one out of four antibodies (NCL-DYS1) showed absence of dystrophin. The protein truncation test detected a truncated dystrophin for both muscle tissue and lymphocytes of this patient next to an additional near normal size fragment in muscle. Genomic sequence analysis revealed a nonsense mutation in exon 29 (4148C > T) of the dystrophin gene. Sequence analysis of the mRNA fragment of the larger peptide showed skipping of exon 29, restoring an open reading frame. Consequently, the epitope of the antibody NCL-DYS1 is mapped to exon 29. The variable clinical features of the three relatives from healthy to severely affected therefore seems to be related to the level of skipping of exon 29. This finding underscores the future potential of gene therapeutic strategies aimed at inducing exon skipping in Duchenne muscular dystrophy, to generate a much milder disease.

Adult↗