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Human Molecular Genetics, 2003, Vol. 12, Review Issue 2 R207-R213
DOI: 10.1093/hmg/ddg260
© 2003 Oxford University Press

Perturbations of chromatin structure in human genetic disease: recent advances

Wendy A. Bickmore1,* and Silvère M. van der Maarel2

1MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, Scotland, UK and 2Department of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarsweg 72, 2333 AL Leiden, The Netherlands

Received June 30, 2003; Accepted July 13, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MUTATIONS IN GENES ENCODING...
 CIS-LINKED ATERATIONS IN...
 REFERENCES
 
Gene expression studies in mammals and simpler eukaryotes have highlighted the central role that chromatin structure and modifications play in both the activation and repression of transcription. Aberrant chromatin structure can cause human genetic disease. Here we discuss recent progress in understanding the molecular mechanisms that underlie three human genetic diseases linked to perturbations of chromatin structure: ICF syndrome, facioscapulohumeral muscular dystrophy and a case of {alpha}-thalassaemia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MUTATIONS IN GENES ENCODING...
 CIS-LINKED ATERATIONS IN...
 REFERENCES
 
It is clear that inappropriate or altered chromatin structure can underlie some human genetic diseases. For example, there are constitutional or acquired mutations in proteins that are involved in DNA methylation and its recognition, the modification of histones or establishing other chromatin states (Table 1) (1). In these cases, genes whose expression is affected are not linked to the underlying mutation. There might also be altered chromatin structure due to mutations linked in cis to the gene(s) whose expression is affected. This could lead to a spreading of ‘silent’ chromatin over neighbouring genes—analogous to classical position-effect variegation (PEV) first documented in Drosophila (2), although clear examples of this mechanism at work in human disease are lacking. Alternatively, there may be mutations in long-range regulatory elements that act to enhance transcription through the formation of specialised chromatin structures (3). Here, we have not tried to present a comprehensive review of chromatin diseases, rather we have highlighted some recent advances in the understanding of such disease mechanisms, and some potentially new mechanisms, by drawing on a few specific examples of human constitutional genetic diseases.


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  		Table 1. Human genetic disorders with mutation in genes encoding chromatin binding proteins or chromatin modifying enzymes
 

   MUTATIONS IN GENES ENCODING CHROMATIN/CHROMATIN MODIFYING PROTEINS
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 ABSTRACT
 INTRODUCTION
 MUTATIONS IN GENES ENCODING...
CIS-LINKED ATERATIONS IN...
 REFERENCES
 
Mutations in several genes that are known to encode proteins that bind to, or modify, chromatin have been identified in human constitutional genetic disease. These are summarized in Table 1, and some of them have been reviewed previously (1). The most recent progress in understanding such a disease state has come from the study of ICF syndrome.

ICF: altered heterochromatin structure, DNA methylation and gene expression
ICF syndrome (OMIM 242860) is a rare recessive disease caused by mutation of the gene encoding a de novo DNA methyltransferase DNMT3B (4,5). ICF syndrome patients display immunodeficiency, facial anomalies, mental retardation and developmental delay. Their lymphocytes show an instability (association, breakage and stretching) of juxtacentromeric heterochromatin of chromosomes 1, 9 and 16 (6), that looks similar to that seen when normal lymphocytes are treated with a DNA methylation inhibitor (7–9). Deletion of the Dnmt3b catalytic domain in mice results in prenatal lethality (10). However, DNMT3b mutations in ICF patients, predominantly missense mutations in the catalytic domain, probably impair, rather than completely abolish, methyltransferase enzyme activity (11–13).

There is not genome-wide hypomethylation in the DNA of ICF patients—the overall levels of 5-methylC are only slightly reduced (14). Rather the hypomethylation is targeted to specific sequences. As expected from the sites of chromosomal instability, there is hypomethylation of satellite DNAs 2 and 3 (15), but in many cell types not just lymphocytes. However, there is also hypomethylation at sequences from chromosomal regions that do not show cytological abnormalities. There have been some reports of undermethylation of {alpha}-satellite DNA and Alu elements, but in a whole-genome approach to identifying sequences that are consistently hypomethylated in lymphoblasts from ICF patients, relative to those of controls, two particular repeats were identified. One of these, D4Z4, is a repetitive sequence implicated in facioscapulohumeral muscular dystrophy (FSHD; see below) (14). Hypomethylation on the inactive X chromosome of female ICF individuals has also been reported (16), but there are variable reports on the effects of this on the escape from X inactivation of X-linked genes in ICF cells (17,18). In addition female ICF patients do not seem to have more severe disease than male patients (17).

Why should particular sequences be hypomethylated in ICF cells? One possibility is that these sequences are present at the chromosomal sites where the DNMT3B enzyme is normally localized. The sub-cellular localization of endogenous human DNMT3B has not yet been reported, but exogenously tagged protein co-localizes with pericentric heterochromatin in some, but not all, murine ES cells (19). The domain necessary for targeting DNMT3B has not been determined, but it is likely to be at the N-terminus where there are two domains commonly found in other chromatin-associated proteins. One of these is a PHD finger similar to that found in ATRX, a protein that also is concentrated at sites of heterochromatin and repetitive DNA sequences (1) (Table 1). There is also hypomethylation of specific repetitive DNA sequences in ATRX patients (but hypermethylation of other sequences) (1). However, there is no chromosome instability reported at the sites of hypomethylation in ATRX cells, and little phenotypic overlap between ATRX and ICF syndromes. The other domain that may be involved in targeting DNMT3B is a PWWP domain. This has been shown to be able to bind to DNA (20), but, based on similarity to tudor and chromo domains, PWWP domains may also bind to methylated proteins (21). A homozygous missense mutation in the PWWP domain has been identified in ICF sibs (22). This serine to proline change occurs within the first {alpha}-helix of a five helix bundle of the PWWP domain (20), and so may have a profound consequence on the mutant protein's structure.

The other possible explanation for the sensitivity of specific repetitive sequences to hypomethylation in the absence of DNMT3b lies in the catalytic properties of the enzyme itself. In vitro the catalytic domain of Dnmt3b shows no preference for the recognition of satellite 2 sequence (12). However, the catalytic domain of Dnmt3b appears to be more processive in its mechanism of action than that of Dnmt3a, and hence in vivo it may be more effective at the methylation of sequences that have a high density of CpGs spread over large genomic regions, as occurs in satellite 2 (12) and D4Z4, which has 290 CpGs in the 3.3 kb repeat unit and a GpC : CpG ratio of 0.8.

Presumably DNA hypomethylation in ICF syndrome leads to dysregulation of genes that perturb cranio-facial, cerebral and immunological development. Recently, microarray analysis was used to identify genes with significantly altered mRNA levels in ICF LCLs as compared with controls (23). Many of the genes identified have known roles in immune function that could account for the immunodeficiency consistently manifest in ICF syndrome. ICF patients generally have normal numbers of B cells, but low serum immunoglobulins (Igs). Decreased levels of Ig heavy chain constant region {gamma} and {alpha} mRNAs but increases of IgHµ and {delta} mRNAs were detected, suggestive of an inhibition of later stages of lymphocyte maturation or activation after V(D)J recombination (23). Nine genes were identified that encode signalling proteins or transcriptional regulators involved in the maturation, migration (RGS1), activation (ID3, lymphocyte-specific TNFRs, TFRC) or homing (integrin ß7) of lymphocytes. Not all of these genes need be direct targets of the DNA hypomethylation and both increases and decreases of specific mRNAs were seen in this experiment. Loss of methylation usually correlates with relief from transcriptional silencing, because methyl-CpG binding proteins (e.g. MeCP2 that is mutated in Rett syndrome; see Table 1) recruit histone deacetylases (HDACs) (24) and histone H3 lysine9 methyltransferases (H3 K9 HMTases) (25) that mediate transcriptional repression. The N-terminal domain of DNMT3B can also recruit HDACs directly (19). Therefore, it is more likely that it is the mRNAs upregulated in ICF lymphoblasts that are the direct targets of hypomethylation. However, no alteration of DNA methylation was detected at the promoters of any of the dysregulated genes tested (23). It is possible that the mRNA for a key master regulator is missing from the chip used in the microarray analysis, but the failure to detect promoter-specific methylation changes in this experiment is consistent with the results of the whole-genome methylation scan (14). That leaves us with the question of how hypomethylation of specific repetitive DNA sequences in ICF patients can lead to altered expression of genes located at distant genomic sites (none of the dysregulated genes are located near to the juxtacentromeric satellite repeats on chromosomes 1, 9 or 16).

One possibility is that the hypomethylation of satellite DNAs alters their properties as heterochromatin, and that it is the physical association of dysregulated genes with these domains in the nucleus that is aberrant in ICF cells, especially lymphocytes. In human and mouse B and T lymphocytes, some silenced genes have been shown to co-localize with domains of pericentromeric heterochromatin (26) and to re-locate away from these domains upon gene activation. In addition, one allele of the IgH genes normally becomes recruited to these domains after V-D-J recombination in activated mature B lymphopcytes (27). It will be of interest to establish the nuclear localization of genes whose expression is dysregulated in ICF syndrome in the B cells of normal and ICF individuals relative to domains of pericentromeric ({alpha}-satellite) and juxtacentromeric (satellites 2 and 3) heterochromatin. Alternatively, the loss of DNA methylation at large arrays of satellite repeats may release repression complexes that are normally bound there to act inappropriately at other genomic sites. Conversely, the expanses of hypomethylated CpGs in the satellite repeats of ICF individuals may sequester protein complexes that normally act to regulate gene expression elsewhere in the genome.


   CIS-LINKED ATERATIONS IN CHROMATIN STRUCTURE
TOP
 ABSTRACT
 INTRODUCTION
 MUTATIONS IN GENES ENCODING...
 CIS-LINKED ATERATIONS IN...
REFERENCES
 
Instances where perturbed gene expression is caused by a chromosome rearrangement in the vicinity of, but outside of, a gene and its promoter generally are classified as ‘position effects’ (3). There are several mechanisms through which gene expression can be altered in these cases. The best understood examples in human disease are those where the rearrangement has separated a gene from its distant regulatory elements, or enhancers (3,28), and these will not be discussed further here. A second mechanism could be the inappropriate spreading of repressive (or active) chromatin across a gene locus by juxtaposition next to specific sequences (e.g. repeats), through the removal of boundary or insulator elements, or through the pairing of heterochromatin and the altered positioning in the nucleus. This mechanism is akin to PEV described in Drosophila (2). A potential example of this mechanism at work in human disease is FSHD

FSHD: gene activation through the loss of repression complexes?
Autosomal dominant FSHD (OMIM 158900), the third most common human myopathy, is mainly characterized by a progressive, and often asymmetric, weakness and wasting of the muscles in the face, shoulder and upper arm. The first symptoms typically present in the second decade of life and additional non-muscular symptoms such as deafness, retinovasculopathy, mental retardation and epilepsy have been recognized as part of the syndrome, mostly in more severely affected patients.

The major locus of this disorder, FSHD1, maps to the subtelomere of chromosome 4q (4qter), where there is a low copy GC-rich repeat consisting of a perfect array of 3.3 kb KpnI units called D4Z4. This polymorphic array may vary between 11 and 150 units in the population while patients show a reduction to 1–10 units as a result of deletion of an integral number of repeat units (29,30). There is an inverse relationship between the residual repeat size and the severity and age at onset of disease. Four to seven repeats are found in the most common form of FSHD, eight to 10 repeats associate with a milder phenotype and reduced penetrance, whereas one to three repeats are found in early onset severe disease (31–33). However, at least one unit of D4Z4 on 4qter is necessary to cause disease since monosomy 4q is not associated with FSHD (34).

Recently it became apparent that a contraction of the D4Z4 repeat per se is not sufficient to cause disease. Distal to D4Z4, two allelic variants of 4qter were identified (called 4qA and 4qB) that, as far as is known, differ by only a few insertion and deletion events (35). However, the 4qA variant does contain regions of ß-(68bpSau3A) satellite repeats distal to D4Z4 that are not present on the 4qB variant (Fig. 1). ß-Satellite is also present at sites of constitutive heterochromatin in the human genome, including pericentric regions of human chromosomes 1 and 9 and the short arms of the acrocentric chromosomes (36). While both 4qA and 4qB variants are almost equally common in the population, FSHD alleles are exclusively of the 4qA-type (37). Further complicating the underlying mechanism, it appears that the subtelomere of 10q (10qter) is an evolutionary duplication of 4qter and so harbors the same highly homologous repeat structure, at the same chromosomal position, as the 4qA variant of 4qter. In fact, 4qter and 10qter are on average 90–95% homologous over a region starting 40 kb proximal to the D4Z4 repeat and extending into the telomere repeat itself (35). Owing to subtelomeric plasticity, exchanges of the repeat structures on both chromosomes are frequently encountered in the population. In some 10% of individuals, one of their chromosomes 10 harbors a 4-type repeat array and a similar proportion of individuals has the opposite constitution with one 10-type repeat on chromosome 4. About half of these exchanged repeat structures is not homogenous but consists of clusters of 4-type and 10-type repeats. Nevertheless, contractions of repeat structure on 10qter, even when they consist of 4-type units, are non-pathogenic. Therefore, two prerequisites are necessary to cause disease: a contraction of the D4Z4 repeat to one to 10 units, and this contraction should occur on a 4qA chromosome.



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  		Figure 1. Alleviation of gene repression through loss of D4Z4 repeats in FSHD. (A) On non-disease (healthy) chromosomes the D4Z4 repeats (black triangles) bind repressor complexes (shaded red circles) that repress the expression of proximal genes on 4q (red box). Upon reduction of D4Z4 repeat number, repressor activity is insufficient to effectively silence these genes resulting in inappropriate gene expression (green box) and FSHD. Gene activation might be aided by proteins bound to the ß-satellite sequences (yellow) distal to D4Z4 on 4qA variant chromosomes. (B) Why are reductions of D4Z4 repeats on 4qB chromosomes that lack ß-satellite, non-pathogenic? Either the loss of D4Z4 repeats leads to loss of repression complexes and upregulation of gene expression, as on 4qA, but for some reason this is insufficient to cause disease, or there is something lacking from 4qB chromosomes (e.g. the ß-satellite) that is necessary to upregulate expression of 4qter genes to pathogenic levels.

 
How does a contracted repeat structure on 4qA chromosomes cause disease? Despite the presence of an open reading frame within D4Z4 encoding a putative homeobox protein called DUX4, expression of this gene has never been observed (38). Therefore the contraction of the D4Z4 repeats probably affects the expression of other genes on 4qter. One hypothesis is that the D4Z4 repeats act as a barrier between the heterochromatic ß-satellite region distal to D4Z4 on 4qA variants, and a more open chromatin structure proximal of D4Z4. Loss of part of the D4Z4 repeat may then allow PEV-like spreading of heterochromatin into the region proximal to D4Z4 thereby shutting down genes within the affected region. However, this scenario seems unlikely given the observation that complete loss of genes from this region (monosomy for 4q) does not result in FSHD (34). Therefore a more plausible hypothesis is that loss of D4Z4 repeats causes the region to adopt a less repressive chromatin structure thereby up-regulating expression from one or more 4qter genes (Fig. 1A).

Evidence in favor of this latter hypothesis was recently put forward. Increased expression of three genes (FRG1 and 2 and ANT1) was detected by RT–PCR from FSHD muscle in comparison to normal muscle (39). In contrast, in the original report describing FRG1, no (allele-specific) transcriptional upregulation was observed in FSHD muscle (40). A protein complex was identified that recognizes a 27 bp element in each D4Z4 unit, and this sequence was shown to have transcriptional repressor activity in reporter assays (39). Three proteins in the complex that binds this element were identified: these are HMG2B, nucleolin and YY1. HMG2B is a chromatin-associated DNA binding protein (Nuclear Protein Database ID=1NP00505; http://npd.hgu.mrc.ac.uk/). Nucleolin is a nucleolar RNA-binding protein involved in ribosome biogenesis (NPD ID=1NP00273), but it can also repress transcription of rRNA genes (41) and relocalizes into the nucleoplasm during stress (42). YY1 (NPD ID=1NP00298) is a homologue of Drosophila pleiohomeotic (pho), the only DNA sequence-specific DNA binding protein in the polycomb silencing complex. Reducing the levels of HMG2B, nucleolin or YY1 in HeLa cells led to increased expression of FRG2 (FRG1 and ANT were not examined), as was seen in FSHD muscle. In cells the protein complex was detected at D4Z4 repeats but not at FRG1 (FRG2 was not tested), whose expression may be affected in FSHD. What could be the mechanism of gene repression in cells with normal numbers of D4Z4 repeats? YY1 can recruit the histone H4 (Arg3) methyltransferase PRMT1 (43) and this (or other) histone modifications could then spread out from D4Z4 to surrounding regions. However, methylation of H4 on Arg3 is usually associated with transcriptional activation (44). The Drosophila YY1 homologue Pho is in the ESC-E(Z) polycomb group complex, which can methylate histone H3 at K27 (45), a modification associated with transcriptional repression (44). Given the high density of methylated CpGs in D4Z4 one might expect there to be MeCP2, or other MBD, associated repression complexes bound there, and their loss when repeats are deleted might also contribute to gene activation on disease chromosomes (24,25). Unlike the situation in ICF syndrome, hypomethylation of the D4Z4 repeats on FSHD disease chromosomes has not been found (46). It will be important therefore to use chromatin immunoprecipitation (ChIP) to analyse the histone modifications, at both the D4Z4 repeats, and the nearby genes. However, the presence of repression complexes at D4Z4 repeats still does not explain why FSHD only occurs on contracted repeats carried on 4qA chromosomes, not on 4qB chromosomes (Fig. 1B), nor why the phenotype is muscle-specific.

If it is hard to explain FSHD through effects purely in cis, could nuclear organization play a role in the pathology of FSHD? Given the presence of nucleolin in the D4Z4-binding complex, it is intriguing that YY1 has been shown to interact with B23/nucleophosmin 1 (47), a protein that is nucleolar in cycling cells, but that locates in the nucleoplasm in G0 cells (NPD ID=1NP00274). A nucleolar connection could explain the association of the FSHD disease phenotype with the ß-satellite containing 4qA variant chromosomes, since the other genomics sites of ß-satellite are associated with the nucleolus (48), and D4Z4 sequences are also present on the acrocentric chromosomes, 1q12, and 10cen interspersed with ß satellite repeats (49). Although no preferential localization of 4qter, from either disease or normal chromosomes, near the nucleolus has been seen in lymphoblasts (50), this issue is worth revisiting in nuclei from non-dividing cells such as muscle. There is an enhanced pairing frequency between subtelomeres of chromosomes 4q and 10q in FSHD patients suggesting that trans-sensing effects might play a role in FSHD pathogenesis (51). Recently, additional evidence supporting a trans effect in FSHD was presented by studing the effect of increasing D4Z4 repeat units on reporter gene activity in murine C2C12 myoblasts (there are no endogenous D4Z4 sequences in the mouse genome). While D4Z4 repeats had only little effect in cis on the transcriptional activity of the reporter gene, myotube formation was strongly impaired (52).

Some genes in 4qter may be upregulated in FSHD as a direct or indirect consequence of the contraction of the D4Z4 repeat. Could there be more? The region proximal to D4Z4 is exceptionally gene-poor with only three identified gene-candidates within an interval of at least 2.5 Mb. However there is a more gene-dense region proximal to this, for which there is little expression data from healthy and control muscle. Extensive expression studies of 4qter genes in appropriate tissue may allow the candidate gene(s) to be prioritized.

Gene inactivation by antisense RNA?
Juxtaposition of repetitive DNA next to genes can lead to gene silencing in many model organisms (2) and, as just discussed, FSHD might be an example of the same mechanism at work in human disease. Could the juxtaposition of transcribed genes also affect the function of neighboring genes? Study of human thalassaemias has been instrumental in elucidating the complex chromatin mechanisms that regulate expression of the {alpha}- and ß-globin genes. Now a case of {alpha}-thalassaemia has highlighted a potentially new mechanism through which genes may be inappropriately silenced—the expression of an aberrant (antisense) transcript from one gene that overlaps the coding region of another (53). In this case a deletion of the 3' portion of the {alpha}-globin locus on chromosome 16p13.3 removes the HBA1 and HBAQ1 genes, but leaves the more 5' HBA2 intact. The deletion also removes the last few exons, and the transcription termination signals, from the widely expressed LUC7L gene that is immediately downstream of the {alpha}-globin complex, and encoded on the opposite strand (Fig. 2A). In this situation HBA2 is not expressed and, moreover, it is methylated (in lymphocytes but not in the germ-line) over a 2 kb region that includes its CpG island. Methylation of {alpha}-globin CpG islands does not normally occur, even in cells that don't express globin genes. The silencing and methylation of HBA2 correlates with the presence of antisense transcripts from the partially deleted LUC7L that, because the signals for normal transcriptional termination have been deleted, extend into HBA2 and its CpG island.



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  		Figure 2. Models for HBA2 silencing by LUC7L antisense transcription. (A) Deletion of 16p13.3 that removes HBA1 and HBQ genes, as well as the 3' end of LUC7L. This results in antisense transcripts from LUC7L extending as far as the CpG island of HBA2. CpG islands of {alpha}-globin genes are normally unmethylated (open lollipops). On the deletion chromosome the HBA2 island is methylated (closed lollipops) (53). (B) Silencing of HBA2 by histone methyltransferases (HMT) that track with the elongating form of R Pol II (blue cylinders) that is phosphorylated at serine 2 of the CTD. In some organisms it is known that histone methylation can lead to DNA methylation (59). (C) Silencing of HBA2 by RNAi. If the aberrant LUC7L antisense transcript is present in a cell that is also transcribing HBA2, then dsRNAs will result. This will trigger the production of siRNAs by the RNAi machinery. In fission yeast and plants, these siRNAs can act as guides to bring RNAi components and associated HMTases to the locus that produced the dsRNA. Since the region of dsRNA encompasses the HBA2 CpG island, H3 at this location will become methylated (on K9) (57), and this in turn may lead to DNA methylation (59) and HBA2 silencing.

 
What are the mechanisms by which an antisense RNA could silence a gene on the opposite strand? Antisense RNA is implicated in the silencing of imprinted genes (54). In the mouse, transcription of non-coding RNA from the Air locus is necessary for the imprinted repression of the overlapping Igf2r on the opposite strand (as well as for that of other non-overlapping genes nearby). It is not clear whether it is the Air transcript itself that is required (perhaps to bind to the chromosome in a manner akin to XIST in X inactivation), or whether it is the act of transcription through the Air locus that is important. The chromatin state of a locus (and hence its activity) can be changed by the passage of an elongating RNA polymerase II (R pol II) through it because histone methyltransferases (HMTases) are recruited to the C terminal domain (CTD) of R pol II when it is phosphorylated at serine 2. This phosphorylated form of R pol II is associated with the processive phases of transcription elongation (55) (Fig. 2B). To date the HMTases associated with elongating R pol II (Set1 and 2 in yeast) catalyse methylation of histone H3 at K4 and 36 respectively. Methylation of H3 K4 is generally thought to mark active chromatin, although Set1 is required for silencing of rDNA genes in yeast, but Set2 is able to repress transcription (44). Hence the passage of an elongating R pol II through a locus could bring about an altered histone methylation pattern. In model organisms it has been shown that histone methylation can direct DNA methylation, and so lock in gene repression. In this model there is no requirement for the production of both HBA2 and LUC7L transcripts in the same cell (Fig. 2B).

An alternative mechanism would be one triggered by RNAi (56). The ability of the RNAi machinery to silence gene expression post-transcriptionally, by degrading mRNAs corresponding to regions of dsRNA, is well understood. In Arabidopsis and fission yeast it has been shown that RNAi can also silence gene expression at the level of transcription by modifying chromatin structure (methylation of histone H3 at K9) at the locus from which the dsRNA originated, and at other homologous loci in trans (57). It is not known whether this pathway of chromatin modification and gene silencing operates in mammalian cells. If it is the underlying mechanism triggering silencing (at least methylation of DNA, since H3 methylation has not yet been analysed) in this recent case of {alpha}-thalassaemia, then there would have to be some stage at which overlapping transcripts from both HBA2 and the aberrant LUC7L were present in the same cell at the same time (Fig. 2C). This is likely to have occurred early in development because {alpha}-globin is expressed in human pre-implantation embryos (58). Whatever the mechanism of gene silencing in this case, unless the promoter of HBA2 is uniquely sensitive to silencing, it raises the possibility that other examples occur in both normal developmental regulation of human genes, and in human disease.


   ACKNOWLEDGEMENTS
 
We thank Nick Gilbert, Nick Hastie and Robin Allshire for critical reading of the manuscript. W.A.B. is funded by the Medical Research Council, UK and the James S. McDonnell Foundation. S.M.v.d.M. is funded 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 (NIH).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 1313322471; Fax: +44 1314678456; Email: w.bickmore{at}hgu.mrc.ac.uk Back


   REFERENCES
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 INTRODUCTION
 MUTATIONS IN GENES ENCODING...
 CIS-LINKED ATERATIONS IN...
 REFERENCES
 

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