Genetic and epigenetic studies of the FSHD-associated D4Z4 repeat

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common hereditary muscle diseases with an estimated frequency of 1 in 20000. The disease has an autosomal dominant inheritance pattern and is characterised by a progressive and often asymmetric muscle weakness with an onset of disease in facial or shoulder girdle muscles. The major locus for FSHD is linked to 4q35, located in the subtelomere on the long arm of chromosome 4. This region harbours a highly polymorphic EcoRI fragment that contains a large polymorphic repeat structure, designated D4Z4, which consists of 3.3 kb tandemly arranged D4Z4 repeat units and is highly susceptible to rearrangement. In the majority of patients this repeat is contracted to an array of 1-10 repeat units. However, 5% of FSHD patients, termed phenotypic FSHD patients, do not manifest a contracted D4Z4 array on chromosome 4, but share all clinical characteristics. Complicating FSHD diagnosis, the subtelomere on the long arm of chromosome 10 (10q26) is highly similar to 4q35 and also contains a nearly identical polymorphic repeat array. However, size reductions of the chromosome 10 repeat array are non-pathogenic. Furthermore, exchanges between arrays on chromosomes 4 and 10 are frequently observed in patients and control individuals, but will only cause disease when a contracted array resides on chromosome 4. Finally, recent analysis of sequences distal to D4Z4 revealed two variants of the 4qter sequence, designated 4qA and 4qB. While both variants are almost equally present in the population, FSHD is uniquely associated with the 4qA variant. Although FSHD is associated with a repeat contraction on 4qA, the exact mechanism causing disease is still unknown. The work in this thesis focused on structure and behaviour of the 4q35 subtelomere, aiming at elucidating possible molecular mechanisms that may mediate or cause FSHD pathology. Subtelomeres are dynamic structures that are more often involved in recombination processes than other parts of the genome. Due to exchanges between subtelomeres of different chromosomes highly homologous DNA sequences can be dispersed throughout pericentromeric and subtelomeric domains in the genome. Since the repeat arrays located on the subtelomeres of the long arms of chromosomes 4 and 10 are highly homologous, we analysed these repeat array configurations in a healthy population, the results of which are presented in Chapters 2and 3. This revealed the existence of translocated repeat arrays in 20% of individuals. Besides the presence of homogeneous chromosome 4 repeat units on chromosome 10, and vice versa, we also detected hybrid arrays that contained repeat units derived from both chromosomes. With regard to repeat array size we observed that on average the arrays on chromosome 4 were longer than those on chromosome 10. This size difference was solely caused by alleles that carried the 4qA variant, as 4qB and 10q alleles did not differ in average length. Both repeat arrays of chromosomes 4 and 10 displayed a similar multimodal allele size distribution that possibly reflects a higher-order chromatin structure. This multimodal distribution on chromosome 4 furthermore indicated the presence of a premutation domain containing alleles shorter than approximately 100 kb. Alleles in this domain may be more prone to contraction to a disease allele than arrays located in the larger repeat size intervals and may thus be predisposing for FSHD development. In Chapters 3 and 4 we examined repeat array configurations in de novo FSHD families and observed somatic mosaicism in more than 40% of cases, in either an FSHD patient or an asymptomatic parent of a non-mosaic patient. This mosaicism for a contracted D4Z4 repeat was more often seen in male than in female patients. In addition, affected females showed a higher proportion of cells with the contracted 4q35 repeat array than males, indicating that females have a higher clinical tolerance for mosaic disease alleles. Consistent with this finding is that, on average, there are more females with mosaicism for the FSHD region among unaffected parents. Besides the observed mosaicism for a contracted FSHD allele generating two genetically distinct cell populations by gene conversion without crossover, we also identified FSHD patients with more complex rearrangements that resulted in three cell populations. This suggests that, alongside gene conversion without crossover, also gene conversion with crossover events that result in contraction and expansion of D4Z4 may contribute to the occurrence of mosaic D4Z4 alleles. Furthermore, whereas we did observe D4Z4 repeat units derived from chromosome 4 on chromosome 10 in mosaic individuals, the reverse configuration was never detected. The presence of such an extra chromosome 4 repeat array may facilitate gene conversion and may thus be a predisposing factor for contraction of the D4Z4 repeat array. Eukaryotic cells have the capacity to epigenetically modify their genomes with a biochemical mark to alter the phenotype without changing the genotype. Two major epigenetic processes that mark chromatin are DNA methylation and the modifications of histones, such as acetylation. Due to the repetitive nature and the location on the subtelomere of 4q35, D4Z4 has been proposed to have a heterochromatic character. Chapters 5, 6 and 7 present data on our studies on the chromatin structure of the D4Z4 repeat array, focussing on a possible change in structure of 4q35 in FSHD patients as a consequence of the chromosomal rearrangement. We demonstrated in Chapters 5 and 6 that the D4Z4 contraction is associated with a significant DNA hypomethylation of D4Z4 at the disease allele in FSHD patients. Furthermore, phenotypic FSHD patients that carry normal-sized 4q35 repeat arrays also showed a pronounced DNA hypomethylation of D4Z4 with levels even below those observed for FSHD patients with a contracted D4Z4 repeat array. While low DNA methylation values in FSHD patients linked to 4q35 are restricted to the disease allele, in phenotypic FSHD patients D4Z4 repeat arrays on both chromosomes 4 were found to be hypomethylated. These findings support an allelespecific chromatin change in FSHD patients with a D4Z4 contraction and strongly suggest that hypomethylation is a key event in the cascade of events causing the FSHD phenotype. Analysis of histone 4 acetylation levels in the 4q35 region, as described in Chapter 7, indicated that the chromatin structure close to D4Z4 resembled that of unexpressed euchromatin rather than constitutive heterochromatin. This suggests that D4Z4 and proximal sequences are not heterochromatic. Contrary to our data presented on D4Z4 methylation, no histone 4 acetylation differences were observed between control individuals and FSHD patients. However, it remains possible that other histone modifications may influence the chromatin conformation upon contraction of the D4Z4 array. The data presented in this thesis challenge the model suggesting the spreading of telomeric heterochromatin in a proximal direction upon contraction of the D4Z4 array, but fit in with other models that have been put forward to explain FSHD pathology. In addition to the repression model, suggesting that upon contraction a local reduction of a specific repressor complex bound to D4Z4 will cause inappropriate activation of 4q35 genes, our data also fit with the looping model, in which communication between a short D4Z4 repeat array and a target gene (or genes) occurs in cis by intrachromosomal looping. The model proposing a heterochromatic chromatin conformation as requirement for proper functioning of D4Z4 remains valid as well. Furthermore, as DUX4 located in D4Z4 encodes a putative homeoboxprotein, we cannot exclude whether expression of DUX4 in early development will be altered due to D4Z4 contraction and so contributes to or even initiates FSHD pathology. Probably the actual disease-causing process will be a combination of the proposed mechanisms in which also the unique perinuclear localisation of chromosome 4q also has to be taken into account. Besides providing new insights in the structure and complex behaviour of the chromosome 4q subtelomere associated with FSHD, this thesis also provides two observations relevant to the clinical practice. First, if mosaicism for D4Z4 is present in the germline, the percentage of these cells carrying the disease allele will determine the risk of having affected offspring, unlike in non-mosaic individuals carrying an FSHD-sized repeat array who have a 50% probability of transmitting the disease. More importantly, since mosaic females can carry a considerably higher percentage of cells with the disease fragment than male mosaics without manifesting FSHD, the disease may more easily go unnoticed in asymptomatic females than previously recognised. This indicates that there might be more apparently healthy women mosaic for a contracted FSHD allele who will have an increased risk of having a child that develops FSHD provided this mosaicism extends to the germline. It is therefore important to screen for mosaicism in de novo FSHD families to provide more accurate information on inheritance risks. Second, the methylation assay described in this thesis may have diagnostic and prognostic value, especially for phenotypic FSHD patients. Since we now know that the level of D4Z4 methylation in these individuals should be below the average methylation observed in FSHD patients with a 4q35 contraction, this can be used as a predictive molecular marker to confirm the status __phenotypic FSHD patient__. Hopefully, these two findings will become implemented in the molecular diagnosis of FSHD and contribute to improved genetic counselling.