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The Rockefeller University Press $30.00J. Cell Biol. Vol. 191 No. 6 10491060
www.jcb.org/cgi/doi/10.1083/jcb.201007028 JCB 104
JCB: Review
Correspondence to Davide Gabellini: gabellini.davide@hsr.it
Abbreviations used in this paper: 3C, chromosome conormation capture; CNV,copy number variation; DRC, D4Z4 repressor complex; FSHD, acioscapulo-humeral muscular dystrophy; H3K9me3, histone H3 lysine 9 tri-methylation;H3K27me3, histone H3 lysine 27 tri-methylation; MAR, matrix attachment re-gion; SNP, single-nucleotide polymorphism.
Copy number variations are an important
source o human genetic diversity
Genetic association studies generally evaluate single-nucleotide
polymorphisms (SNPs), which are single nucleotides at specic
genomic locations that vary between individuals o the same
species. Recent results indicate that the human genome contains
another requent type o polymorphism: copy number varia-
tions (CNVs; Conrad et al., 2010). A CNV is a segment o DNA
that can be ound in various copy numbers in the genomes o
dierent individuals (Fig. 1). CNVs range in size rom a ew
hundred nucleotides to several megabases. Compared with SNPs,
CNVs aect a more signicant raction o the genome and
arise more requently. Hence, CNVs signicantly contribute to
human evolution, genetic diversity, and an increasing number o
phenotypic traits (Stankiewicz and Lupski, 2010).
Depending on the genomic context, CNVs can have vary-
ing eects (Fig. 1). For example, recent data indicate that CNVs
directly alter the structure o 12.5% o protein-coding genes
(Conrad et al., 2010), and there is increasing evidence to suggest
that CNVs play an important role in a number o Mendelian dis-
eases and common complex disorders (Stankiewicz and Lupski,2010). For example, Charcot-Marie-Tooth type 1A disease is
caused by duplications in the PMP22 gene, which encodes an inte-
gral membrane protein that is a major component o compact my-
elin in the peripheral nervous system (Chance et al., 1994). Also,
susceptibility to acquired immune deciency syndrome is aected
by segmental duplications encompassing the CCL3L1 gene,
which encodes the CCR5 chemokine and ligand or the human
immunodeciency virus coreceptor (Gonzalez et al., 2005).
CNVs can also aect gene dosage and expression (Stranger
et al., 2007). Recent data indicate that nonB-DNA orming se-
quences, which are usually enriched in promoter regions, are
also enriched in CNV breakpoints. Thus, the same eatures that
are involved in transcriptional regulation may also be involvedin the ormation o CNVs. As a consequence, CNVs might shape
the evolution o gene regulation (Conrad et al., 2010).
More than hal o the human genome is comprised o repeti-
tive sequences (Neguembor and Gabellini, 2010). Because repeti-
tive sequences can act as substrates or homologous recombination,
their presence acilitates the instability o our genome (Gu et al.,
2008). As a result, repetitive sequences account or a signicant
amount o human CNVs (Warburton et al., 2008).
In this review we will ocus on an important human ge-
netic disease, acioscapulohumeral muscular dystrophy (FSHD),
which is caused by the presence o CNVs o a repetitive sequence
that regulates gene expression.
Clinical eatures o FSHD
FSHD (MIM #158900) is characterized by the progressive
weakness and atrophy o a specic subset o skeletal muscles.
In humans, copy number variations (CNVs) are a com-mon source o phenotypic diversity and disease suscepti-bility. Facioscapulohumeral muscular dystrophy (FSHD) isan important genetic disease caused by CNVs. It is an
autosomal-dominant myopathy caused by a reduction inthe copy number o the D4Z4 macrosatellite repeat lo-cated at chromosome 4q35. Interestingly, the reduction oD4Z4 copy number is not sufcient by itsel to cause FSHD.A number o epigenetic events appear to aect the sever-ity o the disease, its rate o progression, and the distribu-tion o muscle weakness. Indeed, recent fndings suggestthat virtually all levels o epigenetic regulation, rom DNAmethylation to higher order chromosomal architecture,are altered at the disease locus, causing the de-regulationo 4q35 gene expression and ultimately FSHD.
The cell biology o disease
FSHD: copy number variations on the theme omuscular dystrophy
Daphne Selvaggia Cabianca1,2 and Davide Gabellini2,3
1International PhD Program in Cellular and Molecular Biology, Vita-Salute San Raaele University, 20132 Milan, Italy2Division o Regenerative Medicine, San Raaele Scientifc Institute, DIBIT 1, 2A3-49, 20132 Milan, Italy3Dulbecco Telethon Institute, 20132 Milan, Italy
2010 Cabianca and Gabellini This article is distributed under the terms o an AttributionNoncommercialShare AlikeNo Mirror Sites license or the frst six months ater the pub-lication date (see http://www.rupress.org/terms). Ater six months it is available under aCreative Commons License (AttributionNoncommercialShare Alike 3.0 Unported license,as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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onset can vary rom inancy to age 50 (van der Maarel et al.,
2007). Early-onset cases are generally associated with more se-
vere phenotypes (Miura et al., 1998; Klinge et al., 2006). Inter-estingly, the FSHD phenotype is gender dependent. Typically,
males are more severely aected, whereas emales can develop
a milder or asymptomatic orm o the disease (Padberg, 1982;
Zatz et al., 1998; Ricci et al., 1999; Tonini et al., 2004).
Muscle impairment in FSHD is oten asymmetric: mus-
cles on one side o the body appear much more compromised
than on the other side (Kilmer et al., 1995). Various hypotheses
have been proposed to explain this phenomenon, including over-
work weakness and handedness, but the mechanism underlying
this asymmetric phenotype in FSHD remains unknown (Pandya
et al., 2008).
The rate o FSHD progression and the distribution o
muscle weakness are highly variable, even between close amily
relatives. Indeed, these eatures were noted in the rst study
conducted on the disease in the late 1800s (Landoyzy and
Dejerine, 1886). A number o monozygotic twins discordant or
the penetrance o FSHD have been described, pointing to a
strong epigenetic component in the disease (Tawil et al., 1993;
Griggs et al., 1995; Hsu et al., 1997; Tupler et al., 1998).
Although the genetic deect underlying FSHD has been
identied, the molecular mechanism causing the disease re-
mains unclear. Recent results suggest that complex genetic
events contribute to FSHD, as discussed below.
As the name implies, FSHD mostly aects the muscles o the
ace, scapula, and upper arms (Tawil et al., 1998). The peculiar
involvement o specic muscles is such a striking eature oFSHD that it is oten used in the clinic to distinguish FSHD
rom the other orms o muscular dystrophy (Padberg et al.,
1991). FSHD onset generally involves the wasting o acial
muscles, such as orbicularis oris and orbicularis oculi, whereas
others, like the pharyngeal and lingual muscles, are unaected.
As the disease progresses, limb girdle muscles, such as scapula
xator and trapezius, are also aected. Abdominal muscle weak-
ness is another eature o FSHD, causing a characteristic lordotic
posture (an abnormal curvature o the lumbar spine) associated
with a protuberant abdomen. In the most severe cases, the
muscular degeneration can extend to the pelvic girdle and oot
dorsifexor muscles, thereby aecting the ability o the patient
to walk. Approximately 20% o FSHD patients become wheel-
chair bound (Pandya et al., 2008).
FSHD is associated with retinal vasculopathy, a blood vessel
disorder o the retina, in 60% o cases (Tawil and Van Der Maarel,
2006) and sensorineural hearing loss in 75% o aected indi-
viduals (Trevisan et al., 2008). Mental retardation, epilepsy, and
cardiac involvement are also present in FSHD patients more re-
quently than in healthy people (Faustmann et al., 1996; Funakoshi
et al., 1998; Trevisan et al., 2006, 2008; Saito et al., 2007).
Most FSHD patients report their rst symptoms during
the second or third decade o their lie; however, the age o
Figure 1. CNVs and their possible effects on gene expression. CNVs can generate dierent outcomes based on the nature o the aected element. I aCNV encompasses an entire gene(s) locus, the dosage o the gene may be altered, leading to gene amplifcation or deletion. Alternatively, the aectedregion can encompass only part o a gene. In this case, i the CNV includes an exon, it can result in the production o an aberrant protein isoorm. Whena CNV localizes to a nonprotein coding region o the gene it may generate imbalances at the level o splicing or noncoding RNA (ncRNA) production.In addition, extragenic CNVs can give rise to altered gene expression when aecting cis regulatory regions.
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10Copy number variations and FSHD Cabianca and Gabellini
A detailed genomic characterization o the 4q35 region ledto the identication o dierent haplotypes (Fig. 3; van Geel
et al., 2002; Lemmers et al., 2007, 2010a). A simple sequence
length polymorphism is localized 3.5 kb proximal to D4Z4.
D4F104S1 (p13E-11), a region located immediately proximal to
D4Z4, contains 15 SNPs. The most proximal unit o the D4Z4
repeat array contains several SNPs. Finally, a large region o
sequence variation (alleles A, B, or C) has been detected distal
to D4Z4 (Fig. 3). Considering these various eatures, 4q alleles
were subdivided in 18 haplotype variants (Lemmers et al., 2010a).
Importantly, D4Z4 deletions are pathogenic only in a ew o
these haplotype backgrounds (4qA161, 4qA159, and 4qA168;
Lemmers et al., 2007, 2010b). D4Z4 deletions in the presence
o these haplotypes are not sucient to cause FSHD because
4qA161 asymptomatic carriers have been described (Arashiro
et al., 2009), suggesting that these haplotypes represent only a
permissive condition or FSHD rather than being the causative
event. Importantly, it was observed that FSHD2 patients carry at
least one 4qA161 allele (de Gree et al., 2009), urther supporting
the role o this permissive haplotype in the disease.
There are sequences homologous to D4Z4 on several
human chromosomes (Lyle et al., 1995). On chromosome 10q26
there is a repeat array that shares 98% identity with the D4Z4
repeat array at 4q35 (Cacurri et al., 1998). Additionally, high
homology extends to 45 kb proximal o D4Z4 and 1525 kb
distal (van Geel et al., 2002). The 10q and 4q D4Z4 repeats areequally polymorphic, and some individuals have nonstandard,
hybrid alleles containing 4q-derived repeats on chromosome 10
and 10q repeats on chromosome 4 (van Deutekom et al., 1996a;
van Overveld et al., 2000; Lemmers et al., 2010a). Several stud-
ies have reported that the D4Z4 repeats (even i 4q derived) on
10q are not pathogenic, suggesting that 4q-specic sequences
proximal to D4Z4 are required or FSHD (Bakker et al., 1995;
Deidda et al., 1996; van Deutekom et al., 1996a; Lemmers
et al., 1998; van Overveld et al., 2000). By contrast, an excep-
tion to this rule was recently described (Lemmers et al., 2010b).
In this unusual case, only the distal end o the D4Z4 repeat array
FSHD genetics
FSHD is the third most common myopathy, with an incidence
in the general population o 1:15,000 (Flanigan et al., 2001).
The disease is transmitted as an autosomal-dominant character,
although it presents very complex genetics. Up to 30% o cases
are due to de novo mutations (Zatz et al., 1995). Approximately
hal o the de novo cases result rom a post-zygotic mutation
that leads to mosaicism (Griggs et al., 1993; Weienbach et al.,
1993; Upadhyaya et al., 1995; Bakker et al., 1996; van der
Maarel et al., 2000). In more than 95% o cases, the disease
maps to the subtelomeric region o chromosome 4 long arm at
4q35.2 (FSHD1; Wijmenga et al., 1990, 1991). A small percent-
age o FSHD cases are not genetically linked to chromosome 4
(FSHD2; Wijmenga et al., 1991; Gilbert et al., 1992; Bakker
et al., 1995), although no other putative genetic locus has
been identied.
In 4q-associated FSHD cases, the disease is caused by a
molecular rearrangement that causes copy number variations o
a 3.3-kb tandem repeated macrosatellite called D4Z4 (Wijmenga
et al., 1992; van Deutekom et al., 1993). D4Z4 is extremely
polymorphic in the general population (Hewitt et al., 1994;
Winokur et al., 1994), ranging rom 11 to 150 copies. FSHD
patients carry only 1 to 10 units (Fig. 2; Wijmenga et al., 1992;
van Deutekom et al., 1993).
Although FSHD is highly variable, there is a general cor-
relation between the number o residual D4Z4 repeats, the ageo onset, and the severity o the disease (Goto et al., 1995; Lunt
et al., 1995; Zatz et al., 1995; Tawil et al., 1996; Hsu et al.,
1997; Ricci et al., 1999). In particular, larger deletions tend to
be associated with earlier onset and a more rapid progression
o FSHD (Goto et al., 1995; Lunt et al., 1995; Zatz et al., 1995;
Tawil et al., 1996; Hsu et al., 1997; Ricci et al., 1999). Impor-
tantly, it has been reported that at least one copy o D4Z4 is re-
quired to cause FSHD, as individuals with deletions o the entire
repeat array do not display signs o muscular dystrophy (Fig. 2;
Goto et al., 1995; Tupler et al., 1996; Rossi et al., 2007), sug-
gesting that the repeat itsel plays a critical role in the disease.
Figure 2. FSHD is caused by a reduction in the copy number of D4Z4 repeats. (A) Healthy individuals carry 11150 units o D4Z4. (B) FSHD patientshave less than 11 repeats. (C) At least one copy o D4Z4 is required or FSHD development, as individuals completely lacking D4Z4 are healthy.(D) Patients having a large deletion encompassing FRG2 and DUX4chave been described, indicating that these genes are not necessary or FSHD. Dottedlines indicate that distances are not to scale.
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long distance interactions and chromatin loop domain organiza-
tion (Maeda and Karch, 2007). Here, we summarize the studies
that have investigated the epigenetic actors involved in FSHD.
DNA methylation in FSHD. D4Z4 belongs to a
amily o human tandem repeats termed macrosatellites that are
noncentromerically located (Chadwick, 2009). Together with
other members o the amily, such as DXZ4 on chromosome X(Giacalone et al., 1992) and RS447 on 4p (Kogi et al., 1997),
D4Z4 is extremely GC rich.
DNA methylation is a chemical mark added to cytosine
residues by DNA methyltranserases: DNMT1, DNMT3A, and
DNMT3B (Chen and Li, 2004). Mammalian genomes are glob-
ally methylated, with the noticeable exception o short non-
methylated regions called CpG islands. Current data indicate
that promoter methylation leads to stable gene silencing, whereas
intragenic methylation helps to weaken transcriptional noise
(Suzuki and Bird, 2008). In addition, because several transcrip-
tion actors and chromatin-binding proteins, such as CTCF and
YY1, are methylation sensitive (Hark et al., 2000; Kim et al.,
2003), it is clear that DNA methylation can signicantly aect
the occupancy o a specic genomic region.
It has been shown that although D4Z4 is highly methyl-
ated in healthy subjects, FSHD patients have a specic hypo-
methylation o the D4Z4 contracted allele (Fig. 4; van Overveld
et al., 2003; de Gree et al., 2009). Recent ndings indicate that
D4Z4 contraction is always associated with hypomethylation,
irrespective o the chromosome or the haplotype, as deletions
on chromosome 10 and on chromosome 4 in asymptomatic car-
riers are also associated with a reduction in DNA methylation o
the contracted locus (de Gree et al., 2009). Moreover, D4Z4 is
was transerred to chromosome 10. Thus, the 4q35 FSHD can-
didate genes located proximal to the D4Z4 repeat array were
not present on chromosome 10. This nding suggested that
proximal 4q genes are not required or the pathogenesis o
FSHD (Lemmers et al., 2010b). It has to be noted, however, that
this case is a very unusual patient carrying a rare haplotype with
hybrid repeats deleted on 10q chromosome and a permissive4qA161 allele on 4q chromosome (Lemmers et al., 2010b).
Hence, in this case the disease could still be linked to chromo-
some 4 through an in trans eect o the hybrid 10q repeats on
the permissive chromosome 4q.
Although the exact molecular mechanism responsible or
the disease is unknown, it is agreed in the eld that the D4Z4
deletion causes an epigenetic gain-o-unction alteration lead-
ing to the up-regulation o candidate gene(s) (Neguembor and
Gabellini, 2010).
Epigenetic eatures associated with FSHD
Several o the clinical eatures outlined above, such as the gen-
der bias in severity, the asymmetric muscle wasting, and the dis-
cordance in monozygotic twins, suggest that FSHD development
involves epigenetic actors (Neguembor and Gabellini, 2010).
Epigenetic changes do not aect the primary DNA se-
quence; rather, gene expression is altered by changing the con-
ormation o chromatin. Local chromatin structure is regulated
by at least three processes: DNA methylation (Suzuki and Bird,
2008), histone modications (Ruthenburg et al., 2007), and
ATP-dependent chromatin remodeling (Ho and Crabtree, 2010).
In addition, a number o elements (chromatin boundaries, insu-
lators, etc.) aect higher order chromatin structure by regulating
Figure 3. SNPs and sequence variants at 4q35. 18 dierent 4q haplotypes have been described. FSHD patients carry D4Z4 deletions in 4qA161,4qA159, and 4qA168 backgrounds. These genetic contexts represent a permissive condition or the disease rather than a cause given that asymptomaticcarriers have been described. SSLP, simple sequence length polymorphism; TEL, telomere.
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10Copy number variations and FSHD Cabianca and Gabellini
ubiquitination, and SUMOylation o lysine (K) residues,
phosphorylation o serine (S) and threonine (T) residues, methyl-
ation o arginines (R), and ADP-ribosylation o glutamic acid
(Bernstein et al., 2007). Combinations o posttranslational
modications o single histones, single nucleosomes, and
nucleosomal domains establish local and global patterns o
chromatin modications and recruit nuclear actors that medi-
ate downstream unctions (Ruthenburg et al., 2007). These pat-
terns can be altered by multiple extracellular and intracellular
stimuli, and chromatin itsel unctions as a genomic integrator
o various signaling pathways, ultimately aecting cellular
processes such as replication and transcription (Cheung et al.,
2000; Nightingale et al., 2006).
The D4Z4 repeat array appears to be organized in distinct
domains, some characterized by transcriptionally repressive
heterochromatin and others by transcriptionally permissive
euchromatin (Zeng et al., 2009). In particular, on both chromo-
some 4 and 10, the repressive marks o histone H3 lysine 9
tri-methylation (H3K9me3) and histone H3 lysine 27 tri-
methylation (H3K27me3) are both present on some D4Z4 units,
but the permissive mark histone H3 lysine 4 di-methylation is
hypomethylated in patients aected by immunodeciency, cen-
tromeric instability, and acial anomalies syndrome (Kondo
et al., 2000). Interestingly, there are no common traits among
these diseases. These ndings suggest that D4Z4 hypomethyl-
ation is not responsible or FSHD by itsel. Nevertheless, it
is interesting to note that non4q-associated FSHD patients
(FSHD2), which lack D4Z4 contractions, display general D4Z4
hypomethylation on both chromosome 4 alleles and on the two
chromosome 10 alleles (de Gree et al., 2009), pointing to a
general deect in methylation o D4Z4 repeats. Collectively,
these results suggest that D4Z4 hypomethylation might repre-
sent a permissive condition required or FSHD onset or that it
might be a consequence o the primary cause o FSHD.
Histone modifcations in FSHD. Most cellular DNA
is compacted into nucleosomes, in which 146 bp o DNA are
wrapped around a protein octamer composed o two copies o
each core histone H2A, H2B, H3, and H4 (Campos and Reinberg,
2009). Nucleosomes are linked by a variable length o DNA
associated with linker histone H1 (Campos and Reinberg, 2009).
Histone proteins are subjected to dierent posttranslational
covalent modications, including acetylation, methylation,
Figure 4. Epigenetic features at 4q35 in healthy subjects and FSHD patients. In control individuals, the D4Z4 repeat array is characterized by markers ochromatin repression, such as high levels o DNA methylation, SUV39H1-mediated H3K9me3, and EZH2-mediated H3K27me3. In contrast, FSHD patientsdisplay hypomethylation, loss o H3K9me3, and corresponding loss o HP1 and cohesin binding. In healthy subjects, D4Z4 is specifcally bound by EZH2and a repressor complex composed o YY1, HMGB2, and nucleolin. It is still to be determined whether binding o these actors is altered in FSHD patients.The human 4q is perinuclear in both control and FSHD individuals. This localization depends on a region that is proximal to D4Z4 in healthy subjects butis D4Z4-specifc and CTCF-mediated in FSHD patients. A MAR located upstream o the repeat array was ound to be weakened in FSHD, altering the 3Dchromosomal architecture o the region.
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the 4q telomere is located near the nuclear periphery (Masny
et al., 2004; Tam et al., 2004). Interestingly, a sequence 215 kb
proximal to the repeat array shows a stronger localization to the
nuclear rim than D4Z4 in healthy subjects, suggesting that a re-
gion proximal to D4Z4, and not the repeat array itsel, directs
the 4q telomere to the periphery (Fig. 4; Masny et al., 2004).
Recently, Ottaviani et al. (2009b) identied an 80-bp sequence
inside the D4Z4 unit that can trigger perinuclear positioning o
articial telomeres in a CTCF- and lamin Adependent manner
(see below). This property is lost upon D4Z4 multimerization.
Thus, it appears that in healthy subjects, multiple copies o
D4Z4 are located near the nuclear periphery due to a 4q-specic
signal proximal to D4Z4, whereas in FSHD patients the peri-
nuclear location is mediated by D4Z4 (Fig. 4; Ottaviani et al.,
2009b). Although FISH analyses indicate that the peripheral
localization o 4q is maintained in dierent cell types and is
apparently unaltered in FSHD patients compared with controls,
the peripheral environment o the FSHD 4q35 allele may be
altered, and thereby contribute to the aberrant 4q35 gene ex-
pression reported in FSHD (Masny et al., 2004; Tam et al., 2004;
Ottaviani et al., 2009b).In metazoans, the nuclear lamina coats the inner surace o
the nuclear envelope (Hetzer, 2010). Using the DNA adenine
methyltranserase identication approach, lamin-associated do-
mains that correlate with silenced regions have been identied
(Guelen et al., 2008). Intriguingly, a lamin-associated domain has
been mapped to a locus that is 50 kb proximal to D4Z4, which is
consistent with the previous nding that this region has a role in
maintaining gene repression (Guelen et al., 2008). The peripheral
location o 4q seems to be strictly dependent on lamin A, given
that chromosome 4 telomeres are dispersed in cells lacking the
lamin A gene (Masny et al., 2004). Furthermore, chromatin
immunoprecipitation assays revealed that lamin A is associated
with D4Z4 in vivo (Ottaviani et al., 2009a).Altered chromatin organization at 4q35 in
FSHD. As mentioned above, there is no macroscopic relocal-
ization o 4q due to D4Z4 deletion in FSHD. Nonetheless,
more subtle alterations may occur. For example, the 4q35 locus
could be repositioned to a dierent peripheral subdomain, lead-
ing to inappropriate 4q35 gene regulation (Masny et al., 2004;
Ottaviani et al., 2009b). Alternatively, the higher order chroma-
tin structure o the 4q35 locus might be aected. There is grow-
ing evidence to indicate that the three-dimensional organization
o the FSHD region signicantly contributes to the regulation o
gene expression at 4q35 (Petrov et al., 2006; Pirozhkova et al.,
2008; Bodega et al., 2009).
Recently, it has been proposed that the area immediately
proximal to D4Z4 could play a role in FSHD (Lemmers et al.,
2007; Tsumagari et al., 2008). Interestingly, this area has been
suggested to unction as a nuclear matrix attachment region
(MAR; Petrov et al., 2006). Matrix attachment was shown to be
weakened on the contracted chromosome 4 in FSHD-derived
myoblasts compared with controls, leading to a drastic alter-
ation in chromatin loop domain organization (Fig. 4; Petrov
et al., 2006). In particular, whereas a high number o D4Z4 re-
peats maintain the organization o the repeat array and 4q35
genes in two distinct chromatin loops, loosening o the MAR in
present on dierent units (Zeng et al., 2009). Consistent with
previous studies, the authors reported euchromatin histone marks
in the rst proximal D4Z4 unit o the array (Jiang et al., 2003;
Zeng et al., 2009).
The modication o H3K9me3 on D4Z4 is mediated by
the histone methyltranserase SUV39H1 (Zeng et al., 2009).
Interestingly, H3K9me3 is lost in FSHD patients, preventing
the binding o D4Z4 to the heterochromatin-binding protein
HP1 and the sister chromatid cohesion complex, cohesin (Fig. 4).
This loss could lead to the de-repression o 4q35 genes and
muscular dystrophy (Zeng et al., 2009).
In a study aimed at characterizing the chromatin status o
the FSHD region, both D4Z4 and the promoter o the 4q35 gene
FRG1 (see below) were reported to be bound by the transcrip-
tion actor YY1 and the Polycomb Group protein EZH2 (Bodega
et al., 2009). Polycomb Group proteins are chromatin modiers
that implement transcriptional silencing in higher eukaryotes
(Simon and Kingston, 2009). In particular, YY1 and EZH2
binding are reduced both at D4Z4 and FRG1 promoter in myo-
tubes compared with myoblasts (Bodega et al., 2009). As a
consequence, the EZH2-mediated histone repressive markH3K27me3 is also reduced in myotubes compared with myo-
blasts. Accordingly, FRG1 expression is increased in myotubes
compared with myoblasts. Notably, the H3K27me3 modica-
tion at D4Z4 was ound by 3D FISH to be less abundant in
FSHD cells compared with controls (Bodega et al., 2009).
In summary, a number o repressive histone marks are
present on D4Z4 in healthy subjects and their loss in FSHD
might lead to de-repression o 4q35 genes.
A repressor complex binds to D4Z4. A ew years
ago, a 27-bp sequence located inside each D4Z4 unit, termed the
D4Z4 binding element, was identied (Gabellini et al., 2002).
This element is specically bound by a D4Z4 repressor com-
plex (DRC) composed o YY1, HMGB2, and nucleolin (Gabelliniet al., 2002). These actors interact with proteins that mediate
gene silencing and heterochromatin ormation, such as DNA
methyltranserases, histone deacetylases, and HP1 (Ko et al.,
2008; Wu et al., 2009). DRC binds to the 4q35 located D4Z4
in vivo and mediates the transcriptional repression o 4q35 genes
(Gabellini et al., 2002). Thus, the loss o D4Z4 repeats in FSHD
may result in reduced DRC binding to the region and, conse-
quently, reduced silencing o the 4q35 genes (Fig. 4; Gabellini
et al., 2004). Importantly, o the actors that have been identied
to bind D4Z4, YY1 is the only one with sequence specicity.
Thus, it would be interesting to investigate whether the recruit-
ment o actors like SUV39H1, EZH2, HP1, and cohesin to
D4Z4 is mediated by YY1 (Fig. 4).
Subnuclear localization o 4q35. Most nuclear
events do not occur randomly throughout the nucleoplasm;
rather, they are usually limited to specic and spatially dened
sites (Ferrai et al., 2010). Accordingly, the particular intra-
nuclear positioning o a given chromosomal region plays an
important role in several cellular processes, such as transcrip-
tion and replication (Spector, 2001).
Although mammalian telomeres in somatic cells are evenly
dispersed in the inner part o the nucleus (Ludrus et al., 1996;
Nagele et al., 2001; Amrichov et al., 2003; Weierich et al., 2003),
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10Copy number variations and FSHD Cabianca and Gabellini
The 4q35 locus is a relatively gene-poor region (van Geel
et al., 1999; Blair et al., 2002). O the genes that have been
identied at 4q35, those o particular interest areANT1, FRG1,
DUX4c, FRG2, andDUX4 (Li et al., 1989; van Deutekom et al.,
1996b; Gabrils et al., 1999; Rijkers et al., 2004; Bosnakovski
et al., 2008b). We will ocus here on the two main candidate
genes,DUX4 and FRG1. We will not discuss the other genes in
detail because they are less attractive candidates. For example,
DUX4c and FRG2 were ound to be deleted in some FSHD
amilies (Fig. 2; Lemmers et al., 2003), suggesting that they are
not necessary or disease onset. Nevertheless, these genes are
present in most o the aected amilies and it is possible that
they could contribute to the penetrance and severity o the disease.
There is some experimental support or this idea or DUX4c
(Bosnakovski et al., 2008b; Ansseau et al., 2009).
DUX4. Although theDUX4 gene contains an ORF en-
coding a putative double homeobox protein named DUX4, the
D4Z4 repeat was initially considered to be a nonprotein coding
sequence due to lack o evidence o transcription and protein
synthesis. Nonetheless, both DUX4 mRNA and protein were
recently detected in FSHD-derived primary myoblasts but notin controls, suggesting that D4Z4 may directly aect disease
progression through the aberrant production o DUX4 (Dixit
et al., 2007). Because the D4Z4 repeat does not contain a
canonical polyadenylation signal, the mRNA is generated exclu-
sively by transcription o the last, most distal, unit o the array
that extends to a region named pLAM, which contains a poly-
adenylation signal (Dixit et al., 2007). It was recently shown that
the pLAM polyadenylation signal can stabilize DUX4 tran-
scripts that are ectopically expressed in transected C2C12 cells
(Lemmers et al., 2010b). This pLAM sequence also contains a
polymorphism that could aect the polyadenylation o the dis-
talDUX4 transcript (Lemmers et al., 2010b). Because a group
o analyzed FSHD1 patients were all ound to carry the sameSNP in this region, it was suggested that this polymorphism
contributes to the selective stabilization oDUX4 transcripts in
FSHD (Lemmers et al., 2010b). It should be noted, however,
that in this study the permissive 4qA161 allele was compared only
to nonpermissive 4qB alleles or 10qA chromosomes (Lemmers
et al., 2010b). Non-permissive 4qA variants, such as the previ-
ously described 4qA166 (Lemmers et al., 2007; de Gree et al.,
2009), were not analyzed. Hence, the available data do not allow
us to exclude the possibility that the described variations refect
4qA/B or 4q/10q dierences.
TheDUX4 pre-mRNA can be alternatively spliced (Snider
et al., 2009). Interestingly, it was recently reported that muscles
o healthy subjects express low levels o aDUX4 splicing isoorm
encoding or a truncated protein, whereas muscles o FSHD pa-
tients express a splicing isoorm encoding or ull-length DUX4
(Snider et al., 2010). It has also been reported that the repeat array
displays a complex transcriptional prole that includes sense and
antisense transcripts and RNA processing (Snider et al., 2009).
Thus, there may be multiple D4Z4-derived RNA players in FSHD
and uture work will be required to determine their unctions.
Recently, an isogenetic screen to assess the eect o over-
expressing FSHD candidate genesANT1, FRG1, FRG2, DUX4c,
andDUX4 on cell viability was used (Bosnakovski et al., 2008a).
FSHD patients would bring the contracted repeats and 4q35
genes into the same chromatin loop (Petrov et al., 2006). Ulti-
mately, the presence o an enhancer at the 5 end o the D4Z4
unit could cause inappropriate 4q35 gene de-repression in FSHD
(Petrov et al., 2008).
Chromosome conormation capture (3C) is a technique
that identies long distance intra- and inter-chromosomal inter-
actions (Dekker et al., 2002). Using 3C, two groups have inde-
pendently investigated the higher order chromatin organization
o the 4q35 locus (Pirozhkova et al., 2008; Bodega et al., 2009).
Pirozhkova et al. (2008) showed that the telomeric 4qA allele
is in close proximity to the promoters o the 4q35 genes FRG1
and ANT1 in FSHD myoblasts and not in control myoblasts.
The 4qA allele is immediately distal to the D4Z4 repeat array
(Lemmers et al., 2002). Interestingly, an enhancer element was
detected in the 4qA allele that could be involved in the reported
up-regulation oFRG1 and ANT1 in FSHD (Gabellini et al.,
2002; Laoudj-Chenivesse et al., 2005; Pirozhkova et al., 2008).
In the other 3C study, an interaction between D4Z4 and the
FRG1 promoter was identied in human primary myoblasts that
appears to be highly reduced upon myogenic dierentiation(Bodega et al., 2009). Consistent with the observed mis-regulation
oFRG1, a small but statistically signicant reduction in the
D4Z4FRG1 promoter interaction was observed in FSHD myo-
blasts compared with controls (Bodega et al., 2009). Alto-
gether, it appears that in healthy subjects , the FRG1 promoter
is in close proximity with the D4Z4 repeat and the gene is
repressed, whereas in FSHD patients the promoter oFRG1
is in close proximity with the 4qA marker and the gene is up-
regulated (Gabellini et al., 2002; Pirozhkova et al., 2008; Bodega
et al., 2009).
CTCF is a multiunctional DNA-binding protein that is
important or transcriptional regulation, chromatin insula-
tion, and chromatin organization (Filippova, 2008). The same80-bp D4Z4 element mediating the perinuclear positioning
o the 4q telomere is also responsible or the CTCF and A-type
lamin-dependent transcriptional insulator unction o the re-
peat (Ottaviani et al., 2009a). The CTCF binding and insulation
activity are lost upon multimerization o the repeats (Ottaviani
et al., 2009a). As such, it has been proposed that FSHD patients
have a CTCF gain-o-unction phenotype that protects certain
genes rom the infuence o nearby repressive chromatin, ul-
timately generating a 4q35 de-repressed state (Fig. 4; Ottaviani
et al., 2009a).
Clearly, the FSHD locus is organized into a higher order
chromatin structure that undergoes dynamic remodeling. The
3D architecture o the region appears to play a undamental role
in regulating 4q35 chromatin status and gene expression, sug-
gesting that deects in the organization o the epigenome o the
FSHD region could underlie this disease.
FSHD candidate genes
The studies aimed at understanding the molecular basis o FSHD
indicate that an in cis alteration likely leads to the de-repression
o target gene(s). This model provides a valid explanation or
the 4q specicity and the autosomal-dominant transmission o
the disease (Tupler and Gabellini, 2004).
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proteins involved in RNA biogenesis, such as SMN, PABPN1, and
FAM71B (van Koningsbruggen et al., 2007). Interestingly, mu-
tations in SMNand PABPN1 cause myopathies (Calado et al.,
2000; Briese et al., 2005). Together, these results suggest that
FRG1 unctions in RNA processing. Indeed, in FSHD and in
transgenic mice or cells overexpressing FRG1, altered splicing
has been observed or a number o genes (Gabellini et al., 2006;
van Koningsbruggen et al., 2007; Davidovic et al., 2008).
Specic muscle-related unctions or FRG1 have been
identied in dierent animal models (Hanel et al., 2009; Liu
et al., 2010). Interestingly, overexpression oXenopusfrg1 or
C. elegans FRG-1 causes a muscle deect (Hanel et al., 2009;
Liu et al., 2010).
FRG1 contains a single ascin-like domain, a moti that is
associated with actin-bundling properties (Edwards and Bryan,
1995), and it was recently shown that FRG1 can bind F-actin
and promote its bundling (Liu et al., 2010). In agreement with
this nding, in dierent organisms the endogenous FRG1 in
muscle has not only a nuclear distribution but is also a sarco-
meric protein, suggesting that FRG1 might perorm a muscle-
specic unction (Hanel et al., 2010; Liu et al., 2010).FSHD pathology is most prominent in the musculature;
however, up to 75% o FSHD patients display retinal vasculopathy
(Fitzsimons et al., 1987; Padberg et al., 1995). Intriguingly, it
was recently reported that in human tissues the endogenous
FRG1 is strongly expressed in arteries, veins, and capillaries
(Hanel et al., 2010). Moreover, inXenopus FRG1 levels are
crucial or proper vascular development, and up-regulation
o FRG1 leads to a disrupted vascular phenotype (Wuebbles
et al., 2009).
Collectively, these studies indicate that FRG1 overexpres-
sion in dierent animal models is associated with aberrant mus-
cle structure and vasculature, the two most prominent eatures
o FSHD pathology.
Conclusions
Recent studies suggest that copy number variations (CNVs) are
important or human phenotypic diversity and disease suscepti-
bility. DNA repeats account or 55% o the human genome and
a signicant raction o CNVs.
FSHD is an important pathology caused by CNVs o
D4Z4 repeats. It is an extremely complicated and ascinating
disease, and research into this topic is revealing much about the
unctional organization o our genome.
An increasing amount o evidence suggests that the 4q35
macrosatellite repeat D4Z4 plays a crucial role in the chromo-
somal organization o the FSHD region. There is a general con-
sensus that the D4Z4 deletion in FSHD leads to epigenetic
alterations that aect the expression proles o genes within the
FSHD region. Unortunately, despite considerable eort, almost
20 years ater the identication o the genetic deect underlying
the disease, the causative FSHD gene(s) remains unknown, and
no eective treatments or FSHD are currently available.
The heterogeneity in disease maniestation probably re-
fects heterogeneity in gene expression in FSHD. An interesting
possibility, thereore, is that the complexity o FSHD could be
explained by considering it to be a contiguous gene syndrome,
Previous work demonstrated a pro-apoptotic unction or DUX4
(Kowaljow et al., 2007), and DUX4 overexpression was ound to
have a dramatically toxic eect. As a consequence o increased
DUX4 levels, the expression o oxidative stress response genes
and o myogenic actors (such as MyoD and My5) was altered.
Interestingly, a myogenic deect has been reported in FSHD
patients (Winokur et al., 2003a; Celegato et al., 2006).
DUX4 homeodomains are similar to those o Pax3 and
Pax7, which are transcription actors that are pivotal to muscle
unction (Buckingham, 2007). Additionally, the phenotype o
DUX4 overexpression can be rescued by overexpression o Pax3
or Pax7 (Bosnakovski et al., 2008a).
Recently, DUX4 overexpression (even at extremely low
levels) was reported to cause massive apoptosis and severely
abnormal development in Xenopus laevis, a model or verte-
brate development (Wuebbles et al., 2010). Note that an increase
in apoptosis is not generally considered to be a phenotype o
FSHD (Winokur et al., 2003b).
In theory,DUX4 represents an attractive FSHD candidate
gene, as it would easily explain the requirement or at least one
D4Z4 or the development o the disease. Due to its extremetoxicity, however, DUX4 could only unction in FSHD i it is ab-
sent under normal conditions and overexpressed exclusively in the
muscle cell precursors o FSHD patients (Wuebbles et al., 2010).
FRG1. FSHD region gene 1 (FRG1) is highly conserved in
both vertebrates and invertebrates (Grewal et al., 1998). FRG1
is considered a likely candidate gene or mediating FSHD due
to its selective chromosomal location at 4q35, its overexpres-
sion in FSHD samples (Gabellini et al., 2002; Bodega et al.,
2009), and the development o FSHD-like phenotypes ollow-
ing its overexpression in mice,Xenopus laevis, and Caenorhab-
ditis elegans (Gabellini et al., 2006; Hanel et al., 2009; Wuebbles
et al., 2009; Liu et al., 2010). Transgenic mice overexpressing
FRG1 selectively in the skeletal muscle develop pathologieswith physiological, histological, ultrastructural, and molecular
eatures that resemble those o FSHD patients (Gabellini et al.,
2006). Nevertheless, FRG1 overexpression in FSHD patients
is currently controversial, and studies have reported incon-
sistent results (Gabellini et al., 2002, 2006, Dixit et al., 2007,
Osborne et al., 2007; Bodega et al., 2009; Klooster et al., 2009).
The idea that FRG1 plays an important role in FSHD was very
recently challenged by the identication o an unusual FSHD
patient with deletion o D4Z4 only on chromosome 10 (Lemmers
et al., 2010b). However, as stated above, beore discarding a
causative role or FRG1 in FSHD, this patient requires urther
characterization. As discussed below, FRG1 is crucial or proper
muscle unction and vascular development (Gabellini et al.,
2006; Hanel et al., 2009; Wuebbles et al., 2009). Hence, FRG1
could at minimum aect the severity o the disease.
To better understand the role o FRG1 in FSHD, eorts
have been made to characterize its biological unction. The
human endogenous FRG1 protein copuries with the spliceo-
some, the proteinRNA macromolecular complex responsible
or pre-mRNA splicing (Kim et al., 2001; Rappsilber et al.,
2002; Bessonov et al., 2008). When ectopically overexpressed,
FRG1 localizes to nucleoli, Cajal bodies, and speckles (van
Koningsbruggen et al., 2004), and colocalizes and/or interacts with
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where the epigenetic alteration oDUX4, FRG1, and other
potential genes collaborate to determine the nal phenotype.
Finally, because DUX4 behaves as a transcriptional activator
(Dixit et al., 2007), it could play a direct role in transcriptional
overexpression o the other 4q35 genes, providing a uniying
model or the molecular mechanism o the disease.
Due to space limitations, we apologize to our colleagues or the vastly incom-
plete documentation o their important contributions to the topics describedin this review. We thank Drs. F. Jerey Dilworth, Claudia Ghigna, MichaelR. Green, and Lawrence G. Wrabetz or critical reading o the manuscript.
Research in the Gabellini laboratory is made possible by supportprovided by the European Research Council (ERC), the Muscular DystrophyAssociation o the USA (MDA), the European Alternative Splicing Networko Excellence (EURASNET), the Association Francaise contre les Myopathies(AFM), the FSHD Global Research Foundation, a Jaya Motta private donation,and the FSH Society, Inc. Davide Gabellini is a Dulbecco Telethon InstituteAssistant Scientist.
Submitted: 5 July 2010Accepted: 8 November 2010
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