Genetic and epigenetic studies of the FSHD-associated D4Z4 repeat
Overveld, P.G.M. van
Citation
Overveld, P. G. M. van. (2005, April 27). Genetic and epigenetic studies of the
FSHD-associated D4Z4 repeat. Retrieved from https://hdl.handle.net/1887/2310
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in theInstitutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/2310
5
n o n -4 q -lin k e d fa c io s c a p u lo h u m e r a l m u s c u la r
d y s tr o p h y
PGM van Overveld
1, R J F L L em m ers
1, L A S andk u ijl
1,
L E nth o ven
2, S T W ino k u r
3, F B ak els
1, GW Padb erg
4,
G- JB van Om m en
1, R R F rants
1and S M van der
Maarel
1Originally published in:
N ature G enetic s
( 2 0 0 3 ) 3 5 : 3 1 5 - 3 1 7
1 D epartm ent o f H um an G enetic s, C enter fo r H um an and C linic al G enetic s, L eiden U niv ersity M edic al C enter, L eiden, T he N etherlands. 2 D iv isio n o f M edic al P harm ac o lo gy,
L eiden/A m sterdam C enter fo r D rug Researc h, L eiden U niv ersity M edic al C enter, L eiden, T he N etherlands. 3 D epartm ent o f B io lo gic al C hem istry,
C o llege o f M edic ine, U niv ersity o f C alifo rnia, I rv ine C A , U S A .
5.1 Abstract
The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD1, OMIM 158900) is caused by contraction of the D4Z 4 repeat array on 4qter. We show that this contraction causes marked hypomethylation of the contracted D4Z 4 allele in individuals with FSHD1. Individuals with phenotypic FSHD1, who are clinically identical to FSHD1, but have an unaltered D4Z 4, also have hypomethylation of D4Z 4. These results strongly suggest that hypomethylation of D4Z 4 is a key event in the cascade of epigenetic events causing FSHD1.
5.2
Epigenetic changes of DNA have been investigated as causes of monogenic disorders, tumorigenesis and aging and are suspected to be important for common multifactorial diseases also. Monogenic diseases associated with epigenetic phenomena are caused either by mutations in chromatin remodelling factors [1] or by position effect variegation mechanisms mostly involving regulatory elements [19]. An epigenetic role is also prominent in imprinting disorders [16 ].
FSHD1, which progressively and variably affects muscles of the face, shoulder and upper arm [15], has such a suspected epigenetic aetiology. We previously mapped FSHD1 to 4qter and showed that it is caused by contraction of the polymorphic D4Z 4 repeat array [4, 20] (Supplementary Figure 5.1 ). In healthy individuals, D4Z 4 consists of 11-150 units on both chromosomes, whereas individuals with FSHD1 carry one 4q array of 1-10 units. About 5% of individuals with FSHD1 do not have a contraction of D4Z 4 and are considered to have phenotypic FSHD [10].
Several observations suggest an epigenetic aetiology in FSHD1. First, the subtelomere of chromosome 10q contains a nearly identical polymorphic D4Z 4 repeat, but size reductions of this repeat are not pathogenic. Second, exchanges between the homologous repeat arrays on 4q and 10q are frequently observed, but these exchanged repeats are only pathogenic when the contracted form resides on chromosome four [3 ]. Finally, D4Z 4 contractions on 4qter are necessary but not sufficient to cause FSHD1, because the actual pathogenicity is only associated with one of two alleles (4qA) of the 15-20 kb 4qter moiety located immediately distal to D4Z 4 [8].
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unlikely that contraction of the D4Z4 repeat array itself directly results in loss of all or part of a putative gene mutated in FSHD1. Thus, FSHD1 may be due to an epigenetic phenomenon that causes the transcriptional deregulation of genes close to D4Z4.
To explain the epigenetic basis for FSHD1, we examined the DNA methylation, the most common modification of mammalian DNA, known to be involved in development, X-chromosome inactivation, imprinting and gene silencing [18]. We examined two CpG methylation-sensitive restriction sites (BsaAI and FseI) in the first (proximal) unit of the D4Z4 repeat array on chromosome 4q35 (see Supplementary subjec ts and methods and Supplementary Figure 5.1). First, we determined methylation of the proximal D4Z4 unit for the 4q-type repeat exclusively (see examples in Figure 5.1). We then showed by pulsed-field gel electrophoresis that these results were representative for the entire array (Supplementary Figure 5.2).
Non-penetrant gene carriers, clinically unaffected individuals carrying the mutated allele, were similarly hypomethylated at their shortened D4Z4 allele (P < 0.001; Figure 5.2). We found no tissue-specific methylation differences between muscle and blood lymphocytes of three individuals with FSHD1 and three control individuals. Two biopsy samples from clinically affected and unaffected muscle from the same individuals with FSHD1 also show no differences in methylation (data not shown). We also found no effect of age or ageing in lymphocyte DNA from unaffected individuals and controls samples twice with a time interval of several years (data not shown). Thus, D4Z4 methylation seems to be established early and transmitted stably.
In about 5% of affected individuals, FSHD1 shows neither linkage to 4qter nor contraction of D4Z4 and is thought to be caused by defects in other unidentified loci, one of which maps to chromosome 15 [17]. These individuals with phenotypic FSHD1 are clinically indistinguishable from those with 4q-linked FSHD1. Analysis of five individuals with phenotypic FSHD1 showed pronounced hypomethylation for both sites on both chromosomes four (P < 0.001), strongly supporting a central role for D4Z4 hypomethylation in the aetiology of FSHD1 (Figures 5.1 and 5.2 and Supplementary Figure 5.3).
To further determine whether D4Z4 hypomethylation is specific to FSHD1, we analysed DNA of 14 unrelated individuals with muscular dystrophy and of individuals with autosomal recessive immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome. ICF syndrome (OMIM 242860) presents with immunodeficiency, facial anomalies, mental retardation Figure 5.2
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and developmental delay and is caused by mutations in the gene DNA methyltransferase 3B (DNMT3B) [21]. Individuals with ICF have hypomethylation at D4Z4 [7].
Unrelated individuals with muscular dystrophy had methylation comparable to control individuals, but individuals with ICF had even lower methylation than individuals with FSHD1 (P < 0.05 for both sites; Figures 5.1 and 5.2). In contrast to FSHD1, in which hypomethylation is restricted to the disease chromosome, hypomethylation was detected on chromosomes 4q and 10q in ICF (FseI, P < 0.01; BsaAI, P = 0.07 (a trend)).
Recent findings suggest that reduction of a specific repressor complex, associated with D4Z4 contraction, causes improper activation of 4q35 genes in a distance-dependent manner [5]. In contrast, a recent study based on histone H4 acetylation levels suggests that 4qter has properties of unexpressed euchromatin and that a differential long-distance looping mechanism seems to be the probable mechanism [6]. But neither model, spreading or looping, explains the etiological link between 4q allele-specificity of D4Z4 contraction and transcriptional upregulation. Our results showing chromosome-specific hypomethylation may provide this missing link between DNA changes and transcriptional derepression in FSHD1. Whatever the exact nature of this mechanism, our results showing D4Z4 hypomethylation in individuals with 4q-linked and phenotypic FSHD1 strongly supports a central role of D4Z4 demethylation in the pathogenic pathway of FSHD1.
5.3 Supplementary subjects and methods
5.3.1 I ndividuals
All individuals included were previously analysed after informed consent for their D4Z4 allele sizes and constitution of repeat arrays ([11, 12, 14] and unpublished results).
Genomic DNA was isolated from PBLs of 79 control individuals, both family members and unrelated individuals (19 monosomic and 60 disomic), 40 FSHD patients (5 monosomic and 35 disomic), 5 disomic phenotypic FSHD patients, 10 non-penetrant obligate gene carriers (2 monosomic and 8 disomic), 14 individuals with an unrelated muscle disease (BMD (n = 3), CAPN3 (n = 2), DMD (n = 3), DM1 (n = 1), OPMD (n = 3) and !-SARC (n = 2); 2 monosomic and 12 disomic) and 2 disomic ICF patients. DNA was extracted essentially as described by Miller et al.[13].
Kindred 2 is composed of two healthy parents, two healthy daughters, and one affected disomic son who inherited from his mother a 48 kb large 4qA fragment and a 120 kb large 4qB fragment from his father. Finally, family 3 consists of a healthy father and an affected mother and daughter. The mother has a 50 kb large 4qA fragment that is inherited by her affected daughter and a 90 kb large 4qB fragment. The daughter inherited, besides the 50 kb D4Z4
Supplementary Figure 5.1 The D4Z4 methylation assay.
Upper panel (I): Schematic diagram of chromosome 4q35 encompassing the FSHD candidate region. FSHD is caused by contraction of the polymorphic D4Z4 repeat (black arrow heads) to 1-10 units. Also shown are the locations of the ANT1, FRG1, FRG2 and DUX4 genes. The approximate distances to D4Z4 are depicted at the top of the figure. There is also one repeat unit present proximally to the D4Z4 repeat orientated in opposite direction. Lower panel (II): Schematic representation of the modified BglII/BlnI dosage test. By adding different methylation-sensitive enzymes subsequent to incubation with BglII, BlnI and EcoRI, methylation statuses of these sites can be determined. EcoRI does not cleave D4Z4, but is used to fragment the DNA and therefore to increase accessibility of the DNA for these methylation-sensitive enzymes. It cuts outside the BglII restriction sites and is therefore not shown in the overview. Underneath the overview, bars represent the different chromosome 4-derived fragments, which are released after digestion with BsaAI or FseI. FseI will generate a fragment of 3387 bp (b) while BsaAI releases a fragment of 3031 bp (c). All fragments will only be generated when specific CpGs in these restriction sites are not methylated. Besides the chromosome 4-derived fragment of 4061 bp (a), which is not restricted by one of the methylation-sensitive enzymes, the chromosome 10-derived fragment of 1774 bp (d) is shown. Since the restriction sites of both methylation-sensitive enzymes are all located distal to BlnI, any putative methylation of the chromosome 10 is not detected and thus does not interfere in our assay.
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repeat array from her mother, a 132 kb large 4qB fragment from her father. Neither of the maternal grandparents is affected.
5.3.2 M ethylation analysis of proximal chromosome 4 repeat unit
Four µg of PBL-DNA was co-digested with restriction enzymes EcoRI, BglII (MBI Fermentas), and BlnI (Amersham Pharmacia Biotech). Subsequently, 12 units of CpG methylation-sensitive enzyme BsaAI (New England Biolabs Inc.) were added. For CpG methylation-sensitive enzyme FseI, after digestion with EcoRI/BglII/BlnI, five µg of PBL-DNA was precipitated with 1/10 3M NaAc pH 5.3, 2.5" 100% EtOH and the pellet was dissolved in 30 µl Tris/EDTA pH 7.5. Thereafter, an FseI digestion (12 units, New England Biolabs Inc.) was performed. All digestions were incubated overnight according to the manufacturer's instructions.
DNA was separated on a linear 0.8% agarose gel (UltraPURE agarose, GibcoBRL Lifetechnologies Ltd). Electrophoresis was performed in 1" TAE supplemented with ethidium bromide. After electrophoresis, DNA was transferred to a Hybond-XL membrane (Amersham Pharmacia Biotech). The probe p13E-11 (D4F104S1) [20], and an empty vector control probe
were labelled by random priming with 32P-dCTP using the megaprime DNA labelling system
(Amersham Pharmacia Biotech). Hybridisations were performed as described previously [11]. Next, phosphorimager patterns were analysed using ImageQ uant software (Molecular Dynamics). After correction for background, methylation of chromosome 4 alleles was calculated by comparing undigested and digested fragments.
Several control experiments were performed to confirm complete digestion by methylation-sensitive enzymes. Since methylation-insensitive isoschizomers are not available for either of these enzymes, equimolar amounts of a plasmid containing one restriction site for each methylation-sensitive enzyme were added. This allows endogenous verification of complete digestion by hybridisation with an empty vector probe. Although this does not monitor the presence of impurities bound to the genomic DNA, identical results were achieved in the methylation PFGE assays. Moreover, the methylation assay was applied five times independently to DNA of one FSHD patient and two controls without significant differences (P = 0.74; with an average deviation of <3%).
Finally, six FSHD patients and two controls were tested, sampled twice with an interval of at least five years without observing an effect of aging (BsaAI, P = 0.21 and FseI, P = 0.74), and with an average variation in methylation within each individual <2%.
5.3.3 M ethylation analysis of w hole chromosome 4 repeat array
or with EcoRI/BlnI. Subsequently, plugs digested with EcoRI/BlnI were washed in Tris/EDTA pH 7.5 and equilibrated in FseI restriction buffer. Next, 20 units of FseI were added. All digestions were incubated overnight and performed according to the manufacturer's instructions.
Supplementary Figure 5.2
Example of methylation analysis by PFGE of one classical FSHD kindred.
To assess whether the methylation status of the proximal unit reflects that of the entire array, DNA from 15 individuals from 6 FSHD families was digested with EcoRI, BlnI and FseI and separated by pulsed-field gel electrophoresis (PFGE). After hybridisation, extended, regular ladders of digested D4Z4 arrays were detected in the FseI lanes, characteristic for equal partial methylation of all chromosome 4-type repeat units within one array. Moreover, a clear inter-individual and inter-allelic variation of methylation at the FseI site was observed. Demonstrating the hypomethylation of FSHD alleles, the FSHD-specific ladders were far shorter than the control ladders, while also the completely undigested disease fragments typically showed less residual signal intensity in FSHD samples than in normal samples. The four monosomic individuals tested by both pulsed-field and proximal repeat unit tests, showed an average deviation of <5% between both methylation assays.
In this figure, the left panel gives a PFGE example of three individuals: two parents with a de novo FSHD kindred digested with EcoRI/HindIII (“H”), EcoRI/BlnI (“B”) and with EcoRI/BlnI/FseI (“F”), respectively. The father carries three 4-type arrays of 48 kb, 75 kb and 105 kb and one 10-type array of 200 kb (trisomic). The mother carries 4-type arrays of 90 kb and 96 kb and 10-type arrays of 60 kb and 70 kb (disomic). The affected son inherits from the father the 105 kb 4-type and 200 kb 10-type array and from the mother the 60 kb 10-type array and an apparently maternal rearranged FSHD allele of 23 kb (disomic). In the FseI lane, variable ladders of D4Z4 repeat arrays (indicated in white) are hybridising due to the equal partial methylation of each consecutive D4Z4 unit. The marker lane (in kb) is indicated with (M).
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After digestion, DNA was separated by PFGE on a 0.8% agarose gel (MP agarose, Boehringer Mannheim) as described previously [3, 9, 11] with the exception that DNA was transferred to Hybond-XL membranes. Thus, 4q-type alleles were separated from 10q-type alleles by the BlnI sensitivity of the latter [2]. Phosphorimager patterns were again analysed using ImageQuant software.
5.3.4 Statistical analyses
Main effects of age (at time of blood sampling: in years) and gender were assessed with ANOVA. Methylation statuses were compared between healthy family members and healthy unrelated controls by independent sample T-tests. To determine whether methylation status was due to allele constitution, we compared the two most divergent allele constitutions (monosomic patients versus disomic controls) in a non-parametric two independent samples test (Mann-Whitney U test, asymmetric). To determine whether methylation status on either chromosome 4 or 4 and 10 is due to pathology or repeat array constitution, we used independent sample T-tests.
Statistical analyses of all individuals showed no differences in methylation due to age or gender, nor between healthy family members and unrelated controls. In subsequent analyses, all data were pooled across these variables.
The fractions of healthy and disease chromosomes 4 in patients and control individuals were used in a linear regression analysis to determine the contribution of these alleles on percentage of methylation at each restriction site. Monosomic FSHD patients have a fraction of zero (no healthy alleles), disomic patients have a fraction of 1/2 (one FSHD and one healthy allele). Monosomic control individuals have a fraction of 1 (one healthy allele out of one) and disomic
Supplementary Figure 5.3
control individuals have a fraction of 2 (two healthy alleles out of two). The data from monosomic and disomic control individuals were pooled into fraction 1, as both groups only contain healthy alleles.
5.4 Acknowledgements
We dedicate this work to the memory of Lodewijk Sandkuijl, who unexpectedly passed away during the preparation of this manuscript. We thank R ten Hove for contributing to the development of the methylation assays and C Wijmenga and D Smeets for providing material from individuals with ICF. FSHD research is made possible by the Prinses Beatrix Fonds, the Muscular Dystrophy Association USA, the FSH Society, the Stichting FSHD, the Shaw Family and the National Institutes of Health.
5.5 References
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4. Deutekom van JCT, Wijmenga C, Tienhoven van EA, Gruter AM, Hewitt JE, Padberg GW, Ommen van GJ, Hofker MH, and Frants RR (1993) FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit. Hum Mol Genet 2(12): 2037-2042.
5. Gabellini D, Green M, and Tupler R (2002) Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell 110(3): 339-348.
6. Jiang G, Yang F, Overveld van PGM, Vedanarayanan V, Maarel van der SM, and Ehrlich M (2003) Testing the position-effect variegation hypothesis for facioscapulohumeral muscular dystrophy by analysis of histone modification and gene expression in subtelomeric 4q. Hum Mol Genet 12(22): 2909-2921.
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8. Lemmers RJLF, Kievit de P, Sandkuijl L, Padberg GW, Ommen van GJ, Frants RR, and Maarel van der SM (2002) Facioscapulohumeral muscular dystrophy is uniquely associated with one of the two variants of the 4q subtelomere. Nat Genet 32(2): 235-236.
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