Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic repression of the D4Z4 repeat resulting in misexpression of the repeat-encoded DUX4 gene in skeletal muscle. Two... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic repression of the D4Z4 repeat resulting in misexpression of the repeat-encoded DUX4 gene in skeletal muscle. Two mechanisms are known to drive this D4Z4 epigenetic dysregulation: a contraction of the D4Z4 repeat or mutations in DNMT3B or SMCHD1, both epigenetic regulators of the repeat that are responsible for the establishment or maintenance of the repeat’s epigenetic repressive state in somatic cells. However, the aforementioned (epi)genetic changes lead to FSHD only if the individual also has a disease-permissive D4Z4 allele which allows for stable DUX4 expression in skeletal muscle. This disease permissivity of D4Z4 alleles has been attributed to the presence of a DUX4 polyadenylation signal adjacent to the D4Z4 repeat which is used for transcription termination. Despite knowing the root cause of FSHD, to date, there is no curative therapy available for FSHD and in some cases, the genetic etiology of the disease remains unknown. In this thesis, we identified a new FSHD disease gene called LRIF1 and performed its follow-up functional studies in human somatic cells and mouse embryonic stem cells. In addition, we also pursued a new targeted gene therapy for FSHD by employing CRISPR-based mutagenesis of the DUX4 polyadenylation signal. Show less
Epigenetic regulation of gene expression by chromatin modifiers is one of the fundamental cellular processes that allow the different cell types in the body to develop from the totipotent embryonic... Show moreEpigenetic regulation of gene expression by chromatin modifiers is one of the fundamental cellular processes that allow the different cell types in the body to develop from the totipotent embryonic stem cells. However, when this epigenetic control mechanism becomes compromised, such as by mutations in chromatin modifiers, it can lead to the development of disease. An example of such epigenetic disease is facioscapulohumeral muscular dystrophy (FSHD), in which the chromatin structure of the D4Z4 macrosatellite repeat is compromised. The loss of a repressive D4Z4 chromatin structure either by contraction of the repeat to a size of 1-10 D4Z4 units (FSHD1), or by mutations in D4Z4 chromatin repressors such as SMCHD1 (FSHD2), results in inappropriate expression of the DUX4 gene from the repeat in skeletal muscle, which is considered the root cause of FSHD.In FSHD, DUX4 expression causes apoptosis, leading to muscle wasting in the patient. In this thesis, we studied the functionality of SMCHD1, and aimed to understand the DUX4 repressive processes in which SMCHD1 is involved. Furthermore, we gathered information on the different roles that SMCHD1 fulfills, such as X-chromosome inactivation in female cells and telomere maintenance. Show less
In this thesis two diseases that share a common feature of hypomethylation of repetitive DNA are studied: facioscapulohumeral muscular dystrophy (FSHD) and immunodeficiency, centromeric... Show moreIn this thesis two diseases that share a common feature of hypomethylation of repetitive DNA are studied: facioscapulohumeral muscular dystrophy (FSHD) and immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. In FSHD there is hypomethylation of the macrosatellite repeat D4Z4 and the associated DUX4 gene, which is caused by a repeat contraction and/or variants in chromatin modifiers essential for a repressive D4Z4 chromatin structure in somatic cells. In ICF there is hypomethylation of centromeric repeats, which is caused by recessive variants in one of four ICF genes, of which two are established chromatin modifiers. In this thesis, the mutation spectrum of FSHD and ICF has been expanded. The SMCHD1 mutation spectrum in FSHD2 has been expanded with the discovery of exonic SMCHD1 variants, intronic SMCHD1 variants, and whole SMCHD1 gene deletions. In addition, we identified heterozygous variants in a new FSHD2 gene, DNMT3B, in two FSHD2 families. For ICF syndrome we expanded the mutation spectrum in the two most common ICF genes, DNMT3B and ZBTB24. Show less
A large part of the human genome consists of repetitive DNA. In this thesis two human diseases have been studied in which deregulation of repetitive DNA is a central feature: facioscapulohumeral... Show moreA large part of the human genome consists of repetitive DNA. In this thesis two human diseases have been studied in which deregulation of repetitive DNA is a central feature: facioscapulohumeral muscular dystrophy (FSHD) and immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. FSHD is caused by the misexression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded in the D4Z4 repeat array and is silenced in healthy somatic tissues. In this thesis, several aspects of the epigenetic deregulation of DUX4 in FSHD are described. We have analysed possible correlations between disease severity and epigenetic organization of the D4Z4 repeat. Next we showed that cellular ageing results in deregulation of genomic regions like D4Z4. Moreover, we show that SMCHD1 is the main epigenetic repressor of DUX4 in somatic cells. We next showed that DUX4 misexpression results in the activation of an FSHD candidate gene, FRG2. Finally, we report the generation of a transgenic mouse model for FSHD. The disease mechanism of ICF syndrome remains to be elucidated. However, in this thesis we identify two new ICF disease genes. We highlight a role for all four known ICF genes in repressing repetitive DNA, suggesting functional convergence of these genes. Show less
